Method for gene transfer into target cells with retrovirus

ABSTRACT

The present invention provides a method for increasing the efficiency of gene transfer into target cells with a retrovirus. The transduction is affected by infecting target cells with a retrovirus in the presence of a mixture of a functional material having a retrovirus binding domain, and a second functional material having target cell binding domain. The target cells may be selected from the group of unintentional hematopoietic progenitor cells and erythrocyte precursor, specifically pluripotent stem cells or embryopalstic stem cells.

This application is a divisional of Ser. No. 08/809,156, filed on Mar.7, 1997, now U.S. Pat. No. 6,472,204, which is a continuation-in-partapplication of PCT application No. PCT/JP96/03254 filed on Nov. 7, 1996.

FIELD OF THE INVENTION

The present invention relates to a method for increasing the efficiencyof gene transfer into target cells. The method permits efficienttransformation of target cells in various technical fields such asmedical science, cell technology, genetic engineering and developmentaltechnology and a series of techniques relating thereto.

PRIOR ART

Owing to understanding in mechanisms of many human diseases as well asrapid progress in recombinant DNA technology and gene transfertechnology, recently, protocols for somatic gene therapy have beendeveloped for treating severe genetic diseases. In addition, currently,activities have been attempted to apply gene therapy to not onlytreatment of genetic diseases but also treatment of viral infectionssuch as AIDS and cancers.

Almost all the gene transfer experiments in human being heretoforeapproved by Food and Drug Administration (FDA) are transduction of cellsby recombinant retroviral vectors. Retroviral vectors can efficientlytransfer a required exogenous gene into cells to stably integrate theexogenous gene into chromosomal DNA and therefore, especially, they arepreferred gene transfer means for gene therapy wherein long term geneexpression is desired. Such vectors are designed in various ways toavoid any adverse effect on transduced organisms. For example,replication functions of vectors are lost to prevent unlimitedrepetition of infection (transduction) due to auto-replication of thevectors to be used for gene transfer into cells. Since these vectors(replication deficient retroviral vectors) have no capability ofauto-replication, in general, retroviral vectors packaged in viralparticles are prepared by using retrovirus producer cells (packagingcells).

On the other hand, bone marrow cells are a good target for somatic genetherapy because bone marrow cells are easily manipulated in vitro andcontain hematopoietic stem cells capable of auto-replication.Alternatively, human cord blood has previously also been demonstrated tocontain a large number of primitive progenitor cells includinghematopoietic stem cells. When gene therapy is carried out by genetransfer into these target cells and grafting thereof in a living body,the gene thus transferred is expressed over long term in blood cells toeffect lifelong cures for diseases.

However, in spite of intensive studies by various groups, hematopoieticstem cells are one of those whose efficient transduction is difficult.Heretofore, a most efficient gene transfer protocol relating tohematopoietic stem cells of mouse and other animals was co-culture ofhematopoietic stem cells with retrovirus producer cells. However, forclinical gene therapy of human being, cell-free transduction is moredesirable due to concerns about bio-safety. Unfortunately, efficientgene transfer into hematopoietic stem cells has generally not beenpossible without co-culture with retrovirus producer cells.

Recently, it has been reported that the gene transfer efficiency byretroviruses can be improved by a component of an extracellular matrix,fibronectin, or its fragments alone (J. Clin. Invest., 93, pp. 1451-1457(1994); Blood, 88, pp. 855-862 (1996)). In addition, it has also beendisclosed that fibronectin fragments produced by genetic engineeringhave the same properties and, by utilizing them, efficient transfer ofan exogenous gene into hematopoietic stem cells can be carried out (WO95/26200). Binding of a heparin binding domain of fibronectin to aretrovirus is suggested to be concerned in such improvement of the genetransfer efficiency by fibronectin. In all these methods utilizingfibronectin and fibronectin fragments, cells are infected withretroviruses in plates on which fibronectin or its fragment isimmobilized.

OBJECTS OF THE INVENTION

The above-described gene transfer methods utilizing fibronectin andfibronectin fragments are considered to be achieved by fibronectin orits fragment molecules having both retrovirus binding domain and targetcell binding domain on the same molecule (Nature Medicine, 2, pp.876-872 (1996)). Therefore, for efficient gene transfer into varioustarget cells by using the above-described method, it is necessary toprepare a functional material having both virus and target cell bindingdomains on one molecule according to respective particular cells and aproblem still remains in the use thereof as a general gene transfermethod.

Further, the above-described gene transfer method is carried out byimmobilizing fibronectin or a fibronectin fragment on the surface of aplate to be used for culture of target cells upon infection ofretroviruses. However, complicated procedures are required forimmobilization on a plate and this is far from saying a simple andconvenient gene transfer method.

Moreover, the functional material to be used in the above-described genetransfer method is limited to that containing a heparin binding domainderived from fibronectin as a retrovirus binding domain. Then, there arepossibilities that an improved gene transfer method can be developed byfinding out any other retrovirus binding substance.

The object of the present invention is to solve the problem and toprovide a more convenient and efficient gene transfer method.

SUMMARY OF THE INVENTION

The present inventors have found that retrovirus infection by afunctional material, typically, fibronectin or its fragment, can bepromoted, even when a region having a retrovirus binding domain and aregion having a cell binding domain are not present on the samemolecule. That is, the present inventors have found that the efficiencyof gene transfer into target cells by retroviruses can be improved byusing an effective amount of a functional material containing aretrovirus binding domain admixed with a functional material having atarget cell binding domain.

In addition, the present inventors have also found that retrovirusinfection enhancing activity by a functional material can be observedeven when the functional material is not immobilized on a surface of aplate. The present inventors have further found that the efficiency ofgene transfer into target cells can be improved by contactingretroviruses with the target cells in the presence of a functionalmaterial immobilized on beads.

In addition, the present inventors have further found a retrovirusbinding substance which does not contain a heparin binding domainderived from fibronectin and also found that the material andderivatives thereof are useful for gene transfer into target cells withretroviruses. Moreover, the present inventors have succeeded in creationof functional materials useful for gene transfer into target cells withretroviruses. Thus, the present invention has been completed.

Then, the first aspect of the present invention relates to a method forincreasing the efficiency of gene transfer into target cells withretroviruses. The method is directed to transduction of target cellswith a retrovirus and is characterized by infecting the target cellswith the retrovirus in the presence of a mixture of an effective amountof a functional material having retrovirus binding domain, and aneffective amount of another functional material having target cellbinding domain to permit transfer of the gene into the target cells.

The functional material having retrovirus binding domain used in thefirst aspect of the present invention is not specifically limited and,for example, it is a functional material selected from the groupconsisting of the Heparin-II binding domain of fibronectin, a fibroblastgrowth factor, a collagen, a polylysine and functional equivalentsthereof. The functional material having target cell binding domain maybe a substance containing a ligand which can bind to target cells. Asthe ligand, there are cell adhesion proteins, hormones, cytokines,antibodies, sugar chains, carbohydrates and metabolites of target cellsand the like. Examples of adhesion proteins include polypeptides of acell-binding domain of fibronectin. As the cell binding domain offibronectin, there are polypeptides of binding domain to VLA-5 and/orVLA-4. Further, other examples of ligand include erythropoietin.

The functional material to be used in the first aspect of the presentinvention may be used without immobilization or may be immobilized and,when they are immobilized on beads, they can be used conveniently. Inaddition, when a ligand specific for target cells is selected as thefunctional material having target cell binding domain, the first aspectof the present invention permits convenient transduction of intendedtarget cells.

As described above, in the conventional methods as disclosed in WO95/26200 and Nature Medicine, it is consider to be an essentialmechanism for improving the gene transfer efficiency into target cellswith a retrovirus to co-localize the retrovirus and the target cells ona functional material having both retrovirus binding domain and targetcell binding domain on the same molecule. However, according to thepresent invention, the efficiency of gene transfer into target cells canbe improved by carrying out gene transfer into the target cells with aretrovirus in the presence of a mixture of an effective amount of afunctional material having retrovirus binding domain and an effectiveamount of another functional material having target cell binding domain.

The second aspect of the present invention relates to a culture mediumfor target cells to be used for gene transfer into the target cells withretroviruses which comprises a mixture of an effective amount of afunctional material having retrovirus binding domain, and an effectiveamount of another functional material having target cell binding domain.

By using the culture medium of the second aspect of the presentinvention, the first aspect of the present invention can be carried outconveniently.

The third aspect of the present invention relates to a localizationmethod of retroviruses and the method is characterized by incubating aculture medium containing a retrovirus contacted with a mixture of aneffective amount of a functional material having retrovirus bindingdomain, and an effective amount of another functional material havingtarget cell binding domain.

The fourth aspect of the present invention relates to a kit to be usedfor carrying out retrovirus-mediated gene transfer into target cells andthe kit comprises:

(a) an effective amount of a functional material having retrovirusbinding domain and/or an effective amount of another functional materialhaving target cell binding domain;

(b) an artificial substrate for incubating target cells and aretrovirus; and

(c) a target cell growth factor for pre-stimulating the target cells.

By using the reagent kit of the fourth aspect of the present invention,the first and third aspects of the present invention can be carried outconveniently.

The fifth aspect of the present invention relates to a method forimproving the gene transfer efficiency into target cells withretroviruses and the method is characterized by infecting the targetcells with a retrovirus in the presence of an effective amount of afunctional material having a target cell binding domain as well as aretrovirus binding domain derived from a fibroblast growth factor, acollagen or a polylysine, or a functional equivalent thereof on the samemolecule to permit transduction of the target cells.

In the above conventional methods as described in WO 95/26200 and NatureMedicine, fibronectin fragments are disclosed as the material which canbe used in a most efficient method for improving gene transfer intotarget cells with retroviruses. However, regarding functional materialsother than fibronectin fragments, there is no specific disclosure aboutwhat kind of a functional material can be used in an efficient methodfor gene transfer into target cells with retroviruses. Morespecifically, in the conventional method, only the repeat 12-14 offibronectin is disclosed as the retrovirus binding domain.

The present inventors have unexpectedly found that a fibroblast growthfactor, a collagen, a polylysine and so on which do not have anystructural relation to the repeat 12-14 of fibronectin can beeffectively used in a method for improving gene transfer into targetcells with retroviruses. Therefore, any functional equivalent of thesematerials, i.e., any material which has a retrovirus binding domainfunctionally equivalent to these materials and can improve the genetransfer efficiency into target cells with retrovirus can be used in thefifth aspect of the present invention.

In the fifth aspect of the present invention, as the target cell bindingdomain, a material having a ligand which can bind to target cells can beused and this material is coupled to the retrovirus binding domain.

Examples of the ligand include cell adhesion proteins, hormones,cytokines, antibodies, sugar chains, carbohydrates, metabolites oftarget cells and the like. Examples of cell adhesion proteins includepolypeptides of a cell binding domain of fibronectin. For example,polypeptides of binding domain to VLA-5 and/or VLA-4 can be used in thepresent invention. Further, other examples of ligand includeerythropoietin.

In the fifth aspect of the present invention, as the fibroblast growthfactor to be used as the retrovirus binding domain, there are fibroblastgrowth factors selected from, for example, a fibroblast growth factorrepresented by SEQ. ID No. 3 of the Sequence Listing, functionalequivalents of the factor and polypeptides containing the factor orfunctional equivalents thereof.

Examples of these functional materials include polypeptides containingan amino acid sequences represented by SEQ. ID Nos. 4 and 5 of theSequence Listing.

In the fifth aspect of the present invention, collagens to be used asthe retrovirus binding domain include, for example, collagens selectedfrom a collagen fragment containing an insulin binding domain derivedfrom type V collagen, functional equivalents of the fragments andpolypeptides containing the fragments or functional equivalents thereof.In addition, examples of the fragments include a fragment containing anamino acid sequence represented by SEQ. ID No. 6 of the SequenceListing.

Examples of these functional materials include polypeptides representedby SEQ. ID Nos. 7 and 8 of the Sequence Listing.

In the fifth aspect of the present invention, the polylysine to be usedas the retrovirus binding domain is a polymer of L-lysine and, forexample, one having a suitable polymerization degree can be selectedfrom commercially available products and used.

If the retrovirus binding domain derived from a fibroblast growthfactor, a collagen or a polylysine has a target cell binding domain,simultaneously, the gene transfer efficiency into target cells with aretrovirus can be improved by infecting the target cells with theretrovirus in the presence of an effective amount of the retrovirusbinding domain derived from the fibroblast growth factor, the collagenor the polylysine. If target cells are adhesion cells, a retrovirus andtarget cells respectively bind to and adhere to the functional material,and the gene transfer efficiency into the target cells with theretrovirus can be improved by infecting the target cells with theretrovirus in the presence of an effective amount of the retrovirusbinding domain derived from the fibroblast growth factor, the collagenor the polylysine.

It has also been found that, if a polypeptide represented by SEQ. ID No.1 of the Sequence Listing (hereinafter referred to as H-271) has atarget cell binding domain, simultaneously, that is, if target cellsbind to the polypeptide, H-271, the gene transfer efficiency into thetarget cells with a retrovirus can be improved by infecting the targetcells with the retrovirus in the presence of an effective amount of thepolypeptide.

That is, although the retrovirus binding domain disclosed in the aboveNature Medicine is only the repeat 12-14 of fibronectin, the presentinventors have unexpectedly found that this H-271 effectively acts as atarget cell binding domain according to a particular kind of targetcells to improve the gene transfer efficiency into the target cells. Inaddition, in case that target cells are adhesion cells, the target cellsand a retrovirus bind and adhere to the polypeptide, respectively, andthe gene transfer efficiency into the target cells with the retroviruscan be improved by infecting the target cells with the retrovirus in thepresence of an effective amount of the polypeptide.

In the fifth aspect of the present invention, the functional materialmay be used without immobilization or may be immobilized, thoughimmobilization is preferred in case that target cells are adherentcells.

The sixth aspect of the present invention relates to a culture mediumfor target cells to be used for gene transfer into the target cells witha retrovirus which comprises an effective amount of a functionalmaterial which has a target cell binding domain as well as a retrovirusbinding domain derived from a fibroblast growth factor, a collagen or apolylysine, or a functional equivalent thereof on the same molecule.

The seventh aspect of the present invention relates to a localizationmethod of a retrovirus which comprises incubating a culture mediumcontaining the retrovirus contacted with a effective amount of afunctional material containing a retrovirus binding domain derived froma fibroblast growth factor, a collagen or a polylysine. These functionalmaterials can be efficiently used in localization of a retrovirus forimprovement of gene transfer into target cells with the retrovirus.

Moreover, the localization method of a retrovirus of the presentinvention include incubation of the retrovirus contacted with aneffective amount of a functional material comprising a target cell binddomain as well as a retrovirus binding domain derived from a fibroblastgrowth factor, a collagen or a polylysine, or a functional equivalentthereof on the same molecule.

The eighth aspect of the present invention is a kit to be used forcarrying out retrovirus-mediated gene transfer into target cells and thekit comprises:

(a) an effective amount of a functional material having a retrovirusbinding domain as well as a target cell binding domain derived from afibroblast growth factor, a collagen or a polylysine or a functionalequivalent thereof on the same molecule;

(b) an artificial substrate for incubating target cells contacted with aretrovirus; and

(c) a target cell growth factor for pre-stimulating the target cells.

For practicing any method of the first and fifth aspects, any culturemedium of the second and sixth aspects, any method of the third andseventh aspects and any kit of the fourth and eighth aspects of thepresent invention, the functional materials immobilized on beads can besuitably used.

The ninth aspect of the present invention relates to a method forimproving the gene transfer efficiency into target cells with aretrovirus and characterized in that the target cells are infected withthe retrovirus in the presence of an effective amount of a functionalmaterial immobilized on beads selected from substantially purefibronectin, a substantially pure fibronectin fragment or a mixturethereof to permit transduction of the target cells with the retrovirus.

The tenth aspect of the present invention also relates to a method forimproving the gene transfer efficiency into target cells with aretrovirus and characterized in that the target cells are infected withthe retrovirus in the presence of an effective amount of a functionalmaterial selected from substantially pure fibronectin, a substantiallypure fibronectin fragment or a mixture thereof without immobilization topermit transduction of the target cells with the retrovirus.

In the above conventional methods as disclosed in WO 95/26200 and NatureMedicine, it is an essential mechanism for improving the gene transferefficiency with a retrovirus that the retrovirus and the target cellsshould be co-localized on a functional material having a retrovirusbinding domain and a target cell binding domain on the same molecule. Inthese methods, the co-localization of both retrovirus and target cellson the functional material having both retrovirus binding domain andtarget cell binding domain on the same molecule firstly becomes possibleby immobilizing the functional material having the retrovirus bindingdomain and the target cell binding domain on the same molecule on aculture substrate.

However, according to the present invention, even when substantiallypure fibronectin, a substantially pure fibronectin fragment or a mixturethereof is used, unexpectedly, the gene transfer efficiency into targetcells with a retrovirus can be efficiently improved by using thefunctional material having both retrovirus binding domain and targetcell binding domain on the same molecule without immobilization on aculture substrate.

As the target cells to be used in the first, fifth, ninth and tenthaspects of the present invention, there can be used, for example, cellsselected from stem cells, hematopoietic cells, non-adherent low densitymononuclear cells, adherent cells, bone marrow cells, hematopoietic stemcells, peripheral blood stem cells, umbilical blood cells, fetalhematopoietic stem cells, embryoplastic stem cells, embryonic cells,primordial germ cells, oocyte, oogonia, ova, spermatocyte, sperm, CD34+cells, C-kit+cells, multipotential hemopoietic progenitor cells,unintentional hemopoietic progenitor cells, erythrocytic precursorcells, lymphocytic precursor cells, mature blood cells, lymphocytes, Bcells, T cells, fibroblast, neuroblast, nerve cells, endothelial cells,angio-endothelial cells, hepatic cells, myoblast, skeletal muscle cells,smooth muscle cells, cancer cells, myeloma cells and leukemia cells.

As the retrovirus to be used in the first, third, fifth, seventh, ninthand tenth aspects of the present invention, a retrovirus containing anexogenous gene can be used and the retrovirus may be, for example, arecombinant retroviral vector. Further, the retrovirus may be, forexample, a replication deficient recombinant retroviral vector.

The eleventh aspect of the present invention relates to transduced cellsobtained by the first, fifth, ninth or tenth aspect of the presentinvention.

The twelfth aspect of the present invention relates to a cell graftingmethod for grafting the transduced cells of the eleventh aspect of thepresent invention into a vertebrate animal.

The thirteenth aspect of the present invention relates to a polypeptiderepresented by SEQ. ID No. 13 of the Sequence Listing which can improvethe gene transfer efficiency into target cells with a retrovirus, orfunctional equivalents thereof.

The fourteenth aspect of the present invention relates to a geneencoding the polypeptide of the thirteenth aspect of the presentinvention. Examples of the gene include a gene represented by SEQ. IDNo. 17 of the Sequence Listing or a gene which can hybridize to theabove gene under stringent conditions and encode a polypeptide which canimprove the gene transfer efficiency into target cells with aretrovirus.

In the above conventional methods of WO 95/26200 and Nature Medicine,the most efficient peptide to be used for the gene transfer is CH-296.On the other hand, the present inventors have unexpectedly found thatthe same polypeptide without VLA-5 binding domain and VLA-4 bindingdomain can be used in the present invention.

The fifteenth aspect of the present invention relates to a polypeptiderepresented by SEQ. ID No. 30 of the Sequence Listing which can improvea gene transfer efficiency into target cells with a retrovirus, or itsfunctional equivalent.

The sixteenth aspect of the present invention relates to a gene encodingthe polypeptide of the fifteenth aspect of the present invention.Examples of the gene includes a gene represented by SEQ. ID No. 33 ofthe Sequence Listing or a gene which can hybridize to the above geneunder stringent conditions and encode a polypeptide which can improvethe gene transfer efficiency into target cells with a retrovirus.

The seventeenth aspect of the present invention relates to a polypeptiderepresented by SEQ. ID No. 5 which can improve the gene transferefficiency into target cells with a retrovirus, or functionalequivalents thereof.

The eighteenth aspect of the present invention relates to a geneencoding the polypeptide of the seventeenth aspect of the presentinvention. Examples of the gene include a gene represented by SEQ. IDNo. 26 or a gene which can hybridize to the above gene and encode apolypeptide which can improve the gene transfer efficiency into targetcells with a retrovirus.

DETAILED DESCRIPTION OF THE INVENTION

For the gene transfer method of the present invention, usually,recombinant retroviral vectors are used and, in particular, areplication deficient retroviral vector is suitable. The capability ofreplication of the vector is lost to prevent auto-replication ininfected cells, so the vector is non-pathogenic. These vectors caninvade into host cells such as vertebrate animal cells, in particular,mammalian cells to stably integrate exogenous genes inserted into thevectors in chromosomal DNA of host cells.

In the present invention, an exogenous gene to be transferred into cellscan be used by inserting it into a retroviral vector under the controlof a suitable promoter, for example, a promoter of LTR present in aretrovirus or an exogenous promoter. In addition, in order to achievetranscription of an exogenous gene, other regulatory elements which cancooperate with a promoter and a transcription initiation site, forexample, an enhancer, can also be present in a vector. Moreover,preferably, an inserted gene can have a terminator sequence at itsdownstream. The exogenous gene to be transferred into cells can benatural or artificial ones, and can have additional DNA molecule derivedfrom heterologous sources coupled thereto by ligation or other meansknown to the art.

The exogenous genes inserted into a retrovirus can be any genes ofinterest for transduction of the cells. For example, the exogenous genescan encode an enzyme which is associated with a disorder to be treated,a protein, an antisense nucleic acid, a ribozyme or a false primer (see,e.g. WO 90/13641), an intracellular antibody (see, e.g. WO 94/02610), agrowth factor or the like.

The retroviral vectors to be used in the present invention can have amarker gene so that transduced cells can be readily selected. As themarker gene, for example, a drug resistant gene which providestransformant cells with antibiotic resistance or a reporter gene whichprovides transformant cells with an enzyme activity for detectionthereof can be used.

As the vectors to be used, there are retroviral vectors such as knownMFG vector (ATCC No. 68754), α-SGC (ATCC No. 68755) and the like. Inaddition, both N2/ZipTKNEO vector (TKNEO, Blood, Vol. 78, pp. 310-317(1991)) and PM5neo vector (Exp. Hematol., Vol. 23, pp. 630-638 (1995))used in the examples hereinafter contain neomycin resistant genes(neomycin phosphotransferase gene) as their marker genes. Then, cellstransformed with these vectors can be recognized as cells havingresistance to antibiotics (neomycin, G418, etc.) which are inactivatedby the gene products. Moreover, these vectors can be prepared as virusparticles containing the vectors packaged therein by using knownpackaging cell strains, for example, PG13 (ATCC CRL-10686), PG13/LNc8(ATCC CRL-10685), PA317 (ATCC CRL-9078), cell strains described in U.S.Pat. No. 5,278,056, GP+E-86 (ATCC CRL-9642), GP+envAm-12 (ATCC CRL-9641)and the like.

The term “effective amount” of the functional material used herein meansthe amount required for transformation of target cells in gene transferto target cells with a retrovirus. The amount can be selected dependingupon a particular functional material, a retrovirus and a particularkind of target cells by using the method described herein. The term “thegene transfer efficiency” used herein means the transformationefficiency.

The capability of binding to retroviruses of the functional material,i.e., effectiveness and usefulness of the functional material in thepresent invention can be ascertained by routine assays as disclosed inExamples hereinafter.

These assays determine the extent to which retrovirus particles arebound to the functional material immobilized to the matrix to be used inthe present invention so as to resist washing from the matrix. Briefly,for example, a virus-containing supernatant can be incubated in a wellcontaining the immobilized functional material having a retrovirusbinding domain. The well is then thoroughly washed with a physiologicalsaline buffer and thereafter, target cells are incubated in the well todetermine the level of infectious activity of the virus remaining in thewell. The reduction in infectious activity, or titer, relative to theinitial viral supernatant is assessed and compared to that of a similarcontrol (e.g. using a BSA-coated well). A significantly higher titerremaining in the functional material containing well as compared to thecontrol well indicates that the material can be used as the functionalmaterial in the present invention.

To facilitate this screening procedure, the viral vector can contain aselectable marker gene.

The functional material having retrovirus binding domain to be used inthe present invention can be screened by these assays.

As such a functional material having retrovirus binding domain, there isa functional material which has a retrovirus binding domain derived fromHeparin-II binding domain of fironectin, a fibroblast growth factor, acollagen or a polylysine.

The binding of a cell binding domain of the functional material to beused in the present invention to cells, i.e., binding of a materialcontaining a target cell binding ligand to cells can likewise be assayedusing conventional procedures. For example, such procedures includethose described in Nature 352: 438-441 (1991).

Briefly, the functional material having cell binding domain isimmobilized on a culture plate and the cell population to be assayed isoverlaid in a medium, followed by incubation for 30 minutes to 2 hours.After this incubation period, cells non-adherent to the functionalmaterial are retrieved, counted and assayed for viability. Cellsadherent to the functional material are also retrieved using trypsin orcell dissociation buffer (e.g. Gibco), counted and viability tested. Insome cases, for example for hematopoietic colony forming cells, thecells are further cultured for an additional 12 to 14 days to ascertainthe colony forming characteristics of the cells. The percentage ofadherent cells is then calculated and compared to a standard or standardcontrol such as bovine serum albumin (BSA) immobilized on a cultureplate. Substantial binding of the target cells to the assayed functionalsubstance provides an indication that the functional material/cellcombination is suitable for the present invention and the functionalmaterial having cell binding domain can co-exist with or be coupled tothe functional material having retrovirus binding domain, followed byassessing retrovirus infection of the target cells to construct thefunctional material to be used in the present invention.

As the functional material having retrovirus binding domain which can beused in the present invention, as described above, there is a functionalmaterial which has a retrovirus binding domain derived from Heparin-IIbinding domain of fironectin, a fibroblast growth factor, a collagen ora polylysine. All substances which have a retrovirus binding domainequivalent to the above and can improve the gene transfer efficiencyinto target cells with retroviruses by coupling to or co-existing with aligand having target cell binding domain are included in the functionalequivalents to the retrovirus binding domain derived from a fibroblastgrowth factor, a collagen or a polylysine.

The effective amount of the functional material(s) to be used in thepresent invention can be determined by using target cells and aretrovirus in the gene transfer method of the present invention in thepresence of the selected functional material having retrovirus bindingdomain coupled to or coexisting with the functional material havingtarget cell binding domain and assessing improvement of the genetransfer efficiency according to the above-described method.

Hereinafter, the present invention will be illustrated in detail.

One aspect of the present invention is a method for improving the genetransfer efficiency into target cells with a retrovirus. This method ischaracterized by infecting viable target cells with a retrovirus in thepresence of a mixture of the functional material having retrovirusbinding domain and the functional material having target cell bindingdomain which is effective for improving the gene transfer efficiencyinto the target cells with the retrovirus.

This method can be used for obtaining transformant cells transduced withthe retrovirus and grafting the cells into an individual organismpermits gene transfer into an individual organism.

The functional material having retrovirus binding domain to be used inthis method is not specifically limited and examples thereof includeHeparin-II binding domain of fibronectin, a fibroblast growth factor, acollagen, a polylysine and the like. Likewise, functional equivalentsthereof, for example a functional material having a heparin bindingdomain can also be used. In addition, a mixture of the functionalmaterials, a polypeptide containing the functional material, a polymerof the functional material, a derivative of the functional material andthe like can also be used. These functional materials can be obtainedfrom naturally occurring products, or artificially produced (e.g.,produced by genetic engineering techniques or chemical syntheses).Further, they can be produced by combining naturally occurring productswith artificial products.

In so far as the retrovirus binding domain and/or target cell bindingdomain which can achieve gene transfer with the high efficiency asdescribed herein are maintained, the functional material to be used maybe those having mutation in amino acid sequences of naturally occurringpolypeptides. In the present invention, even if deletion, substitution,insertion and/or addition of one or plural, for example, up to severalamino acids are present in the amino acid sequences of naturallyoccurring polypeptides, in so far as the desired retrovirus bindingdomain and/or target cell binding domain are maintained, suchpolypeptides are referred to as functional equivalents of thepolypeptides having naturally occurring amino acid sequences. Thesefunctional equivalents can be obtained by preparing genes encoding thefunctional equivalents to produce the equivalents and ascertaining theirbiological activities.

In this regard, the pertinent biotechnology arts have already advancedto a state in which the deletion, substitution, addition or othermodifications of amino acids in the required functional domains can beroutinely carried out. Then, the resultant amino acid sequences can beroutinely screened for the desired cell binding activity or virusbinding activity.

A gene encoding the functional equivalent can be obtained by searchingfor genes hybridizable to the gene encoding the above functionalmaterial.

That is, the gene encoding the above functional material or a part ofits nucleotide sequence can be used as a probe of hybridization orprimers of a gene amplification method such as PCR or the like to screena gene encoding a protein having a similar activity to the functionalmaterial. Sometimes, in this method, a DNA fragment containing only apart of the desired gene is obtained. In such case, after ascertainingthat the resultant DNA fragment is a part of the desired gene, the wholedesired gene can be obtained by carrying out hybridization with the DNAfragment or a part thereof as a probe or carrying out PCR with primerssynthesized based on the nucleotide sequence of the DNA fragment.

The above hybridization can be carried out, for example, under thefollowing conditions.

That is, a membrane on which DNA is immobilized is incubated in 6×SSC(1×SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% ofSDS, 0.1% of BSA, 0.1% of polyvinyl pyrrolidone, 0.1% of Ficoll 400 and0.01% of denatured salmon sperm DNA together with a probe at 50° C. for12 to 20 hours. After completion of incubation, the membrane is washedwith, firstly, 2×SSC containing 0.5% of SDS at 37° C. and then withchanging the concentrations of SSC to 0.1×SSC and temperatures to 50° C.until the signal derived from the immobilized DNA can be distinguishedfrom the background.

In addition, whether or not the resultant gene thus obtained is thedesired one can be ascertained by examining the activity of the proteinencoded by the resultant gene according to the above method.

As described in the above WO95/26200, Heparin-II binding domain offibronectin is the polypeptide having a retrovirus binding domain.Although a fibroblast growth factor, a collagen and a polylysine do nothave any structural similarity to Heparin-II binding domain offibronectin (e.g., similarity of amino acid sequences), the presentinventors have found that these substances have retrovirus bindingdomains.

The functional material having target cell binding domain to be used inthe present invention is not specifically limited, either, and is asubstance having a ligand which can bind to target cells. Examples ofthe ligand include cell adhesion proteins, hormones, cytokines,antibodies against antigens on cell surfaces, polysaccharides, sugarchains in glycoproteins or glycolipids, metabolites of target cells andlike. In addition, there can be used polypeptides containing thefunctional materials, polymers of the functional materials, derivativesof the functional materials, functional equivalents of the functionalmaterials and the like. These functional materials can be obtained fromnaturally occurring products or artificially produced (e.g., produced bygene engineering techniques or chemical synthetic techniques). Further,they can be produced by combining naturally occurring products withartificially products.

Cell adhesion proteins to be used are, for example, fibronectin and itsfragments. For example, the cell binding domain of human fibronectinwhich corresponds to Pro¹²³⁹-Ser¹⁵¹⁵, as described in U.S. Pat. No.5,198,423, has been shown to have the function equivalent to thepolypeptide C-274 disclosed herein and to bind to cells including BHKand B16-F10 cells (Kimizuka et al., J. Biochem. Vol. 110, pp. 285-291(1991)). A sequence composed of four amino acids of RGDS present inthese polypeptides is a ligand for VLA-5 receptor. Expression of VLA-5receptor is observed in a wide variety of cells and it is expressed inundifferentiated cells better than in differentiated cells. In addition,CS-1 region of fibronectin is known to be a ligand for VLA-4 receptorand binds to cells expressing the receptor (T cells, B cells, monocytes,NK cells, acidophiles, basophiles, thymocytes, myelomonocytic cells,erythroblastic precursor cells, lymphocytic precursor cells, melanoma,muscle cells and the like). The polypeptide described in JP-A 3-284700and represented by SEQ. ID No. 29 (hereinafter referred to as C277-CS1)is a polypeptide having ligands for both above VLA-5 and VLA-4 receptorsand can be used for gene transfer into cells having these receptors.Moreover, it has been shown that Heparin-II domain can bind tofibroblasts, endothelial cells and tumor cells. The polypeptide sequenceof the cell binding domain of Heparin-II domain is useful for directingretrovirus infection toward targeted cells in the presence of apolypeptide of the functional material having retrovirus binding domain.

Hormones and cytokines having cell specific activities are suitable asthe functional material having cell binding domain in the presentinvention. For example, erythropoietin which is a cytokine in thehematopoietic system can be used for gene transfer into erythrocyticcells. Erythropoietin can be prepared according to a known method andused. In addition, functional equivalents of the erythropoietin andpolypeptides containing erythropoietin or functional equivalents thereofcan also be used.

As described in Examples hereinafter, when the functional materialhaving retrovirus binding activity (e.g., H-271 and a fibroblast growthfactor) is used in admixed with C-274 which is a polypeptide having acell binding activity derived from fibronectin or the like, the highgene transfer efficiency can be obtained. NIH/3T3 cells which are usedin these experiments express VLA-5 receptor which can bind to C-274 andthe interaction of them contribute to improvement of the gene transferefficiency.

Further, the same phenomenon is also observed, when an erythropoietinderivative is present in gene transfer into TF-1 cells which expresserythropoietin receptor (Blood, Vol. 73, pp. 375-380 (1989)). Moreover,this effect is not observed in cells which do not have anyerythropoietin receptor.

From these results, it is clear that cell specific increase in the genetransfer efficiency takes place in the presence of the functionalmaterial having retrovirus binding domain together with the functionalmaterial having cell binding domain.

In this aspect of the present invention, the functional material havingretrovirus binding domain is used in the form of a mixture with anotherfunctional material having target cell binding domain. Thereby, the genetransfer efficiency into target cells having affinity to the functionalmaterials is remarkably improved. Since the gene transfer efficiency isimproved, co-culture with virus producer cells can be avoided and thisis one of advantages of the present invention.

Means for selective gene transfer into target cells has high utility andvarious studies have been done. For example, there is non-viral vector(molecular conjugation vector) wherein a material binding to a receptorpresent on a cell surface is coupled to a DNA binding material. Examplesof gene transfer using such a vector include gene transfer into hepatomacells with asialoglycoprotein (J. Biol. Chem., Vol. 262, pp. 4429-4432(1987)), gene transfer into lymphoblasts with transferrin (Proc. Natl.Acad. Sci. USA, Vol. 89, pp. 6099-6103 (1992)), gene transfer intocancer cells with anti EGF receptor antibody (FEBS Letters, Vol. 338,pp. 167-169 (1994)) and the like. These gene transfer methods usingnon-viral vectors are undesirable from the viewpoint of long term geneexpression of transferred genes because the transferred genes are notintegrated into chromosomal DNA of cells. Activities have been attemptedto use retroviruses which are widely used as vectors capable ofinsertion of genes into chromosomes to infect specific cells. Forexamples, gene transfer into hepatocytes by direct chemical modificationof retroviruses to couple to lactose (J. Biol. Chem., Vol. 266, pp.14143-14146 (1991)), gene transfer into erythropoietinreceptor-expressing cells by utilizing recombinant viral particleshaving an envelope protein which is a fused protein with erythropoietin(Science, Vol. 266, pp. 1373-1376 (1994)) and the like have beendeveloped. However, for this purpose, it is necessary to prepare specialprotein particles according to particular target cells. In addition,chemical modification of viral particles requires complicated proceduresand is liable to inactivate viruses. Moreover, regarding a virusenvelope modified by gene engineering, the desired product havingrequired functions (binding to target cells and construction of viralparticles) is not always obtained.

The above WO 95/26200 suggests that a retroviral vector without anyspecial modification can be transferred into cells in the presence of afibronectin fragment to which a suitable ligand having cell bindingactivity is covalently coupled. However, this method uses a functionalmolecule having both virus binding activity and cell binding activityand therefore an individual special functional material should beprepared according to particular kinds of cells. In addition, it isunknown whether or not the functional material prepared maintains bothactivities.

The combination of the functional material having retrovirus bindingdomain and the different functional material having target cell bindingdomain of the present invention can provide a gene delivery system usingretroviruses for a wide variety of cell species. For this purpose, thefunctional material having retrovirus binding domain does not need to becovalently coupled to the functional material having cell bindingdomain. Therefore, there is no need to prepare an individual specialfunctional material wherein the functional material having retrovirusbinding domain is covalently coupled to the functional material havingcell binding domain according to particular kinds of cells and genetransfer into target cells can be conveniently and efficiently carriedout.

Examples of gene transfer into target cells using the method of thepresent invention is gene transfer into cells of the hematopoieticsystem. It has been known that the above CS-1 cell adhesion region offibronectin is useful for gene transfer into hematopoietic stem cells.Further, it has also been known that, in addition to the aboveerythropoietin, various other cell specific cytokines are concerned indifferentiation of hematopoietic cells, and gene transfer can be carriedout specifically into target cells (cell lines) by utilizing them. Forexample, when G-CSF is used, megakaryoblasts and granulocytic precursorcells can be used as the target cells of transduction.

When using a substance which specifically or predominantly binds tomalignant cells as the functional material having cell binding domain,gene transfer into such target cells can be carried out.

For example, it has been known that receptors named as HER-2 and HER-4are expressed in certain breast carcinoma cells (Proc. Nat. Acad. Sci.USA, Vol. 92, pp. 9747-9751 (1995)). Accordingly, it is possible tocontrol growth of breast carcinoma cells by combining heregulin which isa ligand for the receptors with the functional material havingretrovirus binding domain.

In addition, by using the functional material containing iodine forthyroid (cancer) cells or the functional material containing ahigh-density lipoprotein (HDL), an asialoglycoprotein or a part thereoffor liver (cancer) cells, these cells can be used as the target cellsfor transduction.

Further, by using antibodies against antigens present on cell surfaces,suitably, monoclonal antibodies as the functional material having cellbinding activity, any cells whose antibodies are available can be usedas target cells. Thus, a wide variety of cells can be used as the targetcells by utilizing the localization method of a retroviral vector andtarget cells disclosed by the present invention.

In the particularly preferred aspect, the gene transfer efficiency intotarget cells with a retrovirus is increased by using a novel functionalmaterial.

Heretofore, only Heparin-II domain of fibronectin has been known to bethe functional material having retrovirus binding domain effective forgene transfer into target cells with retroviruses.

As described above, the domain itself has that binding to certain cellsand, in some cases, this activity is undesired depending upon certaintarget cells. In such cases, the desired results can be obtained byreplacing the binding domain with another cell binding domain. In thismanner, plural functional materials having different properties can beused and this makes broader application of the gene therapy according tothe present invention possible and transduction of the intended targetcells can be readily carried out.

The novel functional material having retrovirus binding domain providedby the present invention include fibroblast growth factors, polypeptidescontaining the factors, collagen fragments, a mixture of the fragments,polypeptides containing the fragments, polymers of the functionalmaterial and the like. Polylysines are also used for this purpose of thepresent invention. These functional materials can be obtained fromnaturally occurring products or can be artificially produced (e.g.,produced by genetic engineering techniques or chemical syntheses).Further, they can be produced by combination of naturally occurringproducts and chemically synthesized products. The function material canbe used for the gene transfer method of the first aspect of the presentinvention and chimera molecules of the functional material and the otherfunctional material having cell binding domain are also useful for genetransfer.

All the above-described functional materials have retrovirus bindingactivity. However, these materials do not contain Heparin-II domain ofhuman fibronectin described in WO 95/26200 or polypeptides havingsimilar amino acid sequences.

As the fibroblast growth factor, a substantially purified naturallyoccurring product can be used or a product prepared by geneticengineering techniques can be used. In the present invention, thefibroblast growth factor, that is represented by SEQ. ID No. 3 of theSequence Listing can be used and modified derivatives thereofmaintaining functions of the polypeptide can also be used. Examples offibroblast growth factor derivatives include a polypeptide representedby SEQ. ID No. 4 of the Sequence Listing (hereinafter referred to asC-FGF•A). This is a polypeptide wherein the cell adhesion domainpolypeptide of fibronectin is coupled to the N-terminal of a fibroblastgrowth factor represented by SEQ. ID No. 3 and can be produced bygenetic engineering techniques as generally disclosed in U.S. Pat. No.5,302,701. The polypeptide can be obtained by using E. coli which hasbeen disclosed in the above U.S. patent as FERM P-12637, and nowdeposited under Budapest Treaty with National Institute of Bioscienceand Human-Technology (NIBH), Agency of Industrial Science & Technology,Ministry of International Trade & Industry of 1-1-3, Higashi,Tsukuba-shi, Ibaraki-ken, Japan, under the accession number of FERMBP-5278 (date of original deposit; Dec. 9, 1991).

A polypeptide derivative of the above C-FGF•A having CS-1 cell adhesiondomain derived from fibronectin which is represented by SEQ. ID No. 5(hereinafter referred to as C-FGF-CS1) can be obtained by usingEscherichia coli deposited under Budapest Treaty with the NIBH of 1-1-3,Higashi, Tsukuba-shi, Ibaraki-ken, Japan, under the accession number ofFERM BP-5654 (date of original deposit: Sep. 6, 1996) according theprocess described herein. This C-FGF-CS1 is particularly useful for genetransfer into target cells having CS-1 binding property, in particular,hematopoietic stem cells.

As collagen fragments, substantially purified fragments obtained byenzymatically or chemically cleaving natural collagens can be used orthose prepared by genetic engineering techniques can be used. Inaddition, modifications of these fragments maintaining their functionscan be used. Among collagens, human type V collagen has strong insulinbinding activity (JP-A 2-209899). An example of polypeptides havinginsulin binding domain is a polypeptide which contains an amino acidsequence represented by SEQ. ID No. 28 of the Sequence Listing (JP-A5-97698), for example, a polypeptide represented by SEQ. ID No. 6 of theSequence Listing (hereinafter referred to as ColV). ColV can be preparedaccording to a method disclosed in Examples herein. A polypeptide whichcontains ColV and represented by SEQ. ID No. 7 (hereinafter referred toas C277-ColV) is the polypeptide wherein the cell adhesion domainpolypeptide of fibronectin is coupled to the N-terminal of ColV and canbe produced by genetic engineering technique according to JP-A 5-97698as described above. C277-ColV can be obtained by using E. coli which isdisclosed under the accession number of FERM P-12560 in JP-A 5-97698 anddeposited under Budapest Treaty with NIBH of 1-1-3, Higashi,Tsukuba-shi, Ibaraki-ken, Japan, under the accession number of FERMBP-5277 (date of original deposit: Oct. 7, 1991).

A polypeptide (hereinafter referred to as C-ColV-CS1) derived fromC277-ColV which is represented by SEQ. ID No. 8 and has CS-1 celladhesion domain derived from fibronectin can be prepared as follows. ADNA fragment is isolated by amplifying by PCR using the above plasmidpCH102 which is prepared from E. coli deposited under Budapest Treatywith NIBH of 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan, under theaccession number of FERM BP-2800 (date of original deposit: May 12,1989) as a template and the primers CS1-S (the nucleotide sequence isrepresented by SEQ. ID No. 9 of the Sequence Listing) and M4, and thendigesting with the restriction enzymes NheI and SalI.

On the other hand, a DNA fragment is isolated by amplifying by PCR usingthe plasmid pTF7520ColV, which contains a gene encoding C277-ColV andprepared from above E. coli FERM BP-5277 as a template and the primersCF and CNR, and then digesting with the restriction enzymes AccIII andNheI. The nucleotide sequences of CF and CNR are represented by SEQ. IDNos. 10 and 12 of the Sequence Listing. The above two DNA fragments aremixed and ligated with an about 4.4 kb DNA fragment obtained bydigesting the plasmid pTF7520ColV with the restriction enzymes AccIIIand SalI. The resultant plasmid encodes the polypeptide C-ColV-CS1 whichhas CS-1 cell adhesion domain at the C-terminal of C277-ColV and inwhich the second glutamic acid from the C-terminal of ColV and theC-terminal threonine are replaced by alanine and serine, respectively.After culture of E. coli transformed with this plasmid, the desiredpolypeptide can be obtained from the culture. This C-ColV-CS1 isparticularly useful in gene transfer into a target cell having CS1binding property, especially, stem cells.

As the polylysine, as described above, that having a suitablepolymerization degree can be selected from commercially availablepolylysines and used.

The functional materials to be used in the present invention can includederivatives of the above functional materials. Examples thereof includethe above C-FGF-CS1 or its functional equivalents and C-ColV-CS1 or itsfunctional equivalents. In addition, polymers obtained by polymerizingplural molecules of the functional materials and modified materialsobtained by modifying the functional materials according to knownmethods (addition of sugar chain, etc.) can also be used in the presentinvention. These polymers and their functional equivalents can beprepared by genetic engineering techniques using genes encoding thepolymers and genes encoding their functional equivalents. In addition, acysteine-added functional material useful for preparing a polymer of thefunctional material can be prepared by addition, insertion andsubstitution of cysteine in the amino acid sequence of the functionalmaterial. In addition, a molecule which is a cysteine-added functionalmaterial and has a retrovirus binding domain is readily coupled to ananother molecule which is a cysteine-added functional material and has atarget cell binding domain. Furthermore, a material coupled to otherfunctional material can be prepared by utilizing the reactivity of thecysteine residue of the cysteine-added functional material.

In another preferred aspect of the present invention, gene transfer iscarried out by using a polymer of the retrovirus binding domain offibronectin which increases the gene transfer efficiency into targetcells with retroviruses.

The functional material is a polypeptide having plural Heparin-IIbinding domains of human fibronectin in one molecule as described in theabove WO 95/26200 or derivatives of the polypeptide. In so far as thesame activity as that of the functional material is maintained,functional equivalents a part of whose amino acid sequences aredifferent from that of the naturally occurring products can be included.

Examples of the polymer of the functional material include thoseobtained by enzymologically or chemically polymerizing the abovepolypeptide derived from fibronectin or by gene engineering techniques.An example of a polypeptide which has two Heparin-II binding domainsderived from fibronectin in a molecule include a polypeptide having anamino acid sequence represented by SEQ. ID No. 13 of the SequenceListing (hereinafter referred to as H2-547). H2-547 can be obtainedaccording to the method described herein by using E. coli which has beendeposited under Budapest Treaty with NIBH of 1-1-3, Higashi,Tsukuba-shi, Ibaraki-ken, Japan, under the accession number of FERMBP-5656 (date of original deposit: Sep. 6, 1996). A polypeptide havingan amino acid sequence represented by SEQ. ID No. 14 of the SequenceListing is a polypeptide derivative containing a cell adhesionpolypeptide of fibronectin coupled at the N-terminal of H2-547(hereinafter referred to as CH 2-826). This polypeptide can be obtainedaccording to the method disclosed herein. Further, a polypeptide havingan amino acid sequence represented by SEQ. ID No. 30 of the SequenceListing is a polypeptide derivative containing CS-1 cell adhesion regionof fibronectin coupled at the C-terminal of H2-547 (hereinafter referredto as H2S-573). The polypeptide can be obtained according to the methoddescribed herein by using E. coli which has been deposited underBudapest Treaty with NIBH of 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken,Japan, under the accession number of FERM BP-5655 (date of originaldeposit: Sep. 6, 1996). H2S-573 having CS-1 cell adhesion region isuseful for gene transfer into hematopoietic stem cells.

In yet another preferred aspect of the present invention, viable targetcells are infected with a replication deficient retroviral vector in thepresence of the functional material immobilized on beads which iseffective to increase the gene transfer efficiency into cells with aretroviral vector.

Conventional methods for improving the gene transfer efficiency intotarget cells with a retroviral vector by using the functional materialsdescribed in the above WO 95/26200 and Nature Medicine are carried outby immobilizing the functional materials on a vessel to be used forinfection of cells with viruses (a plate for cell culture). Thesemethods require complicated procedures such as washing of excessfunctional material after treatment of the plate with a solutioncontaining the functional material.

Then, the gene transfer methods using a plate having the functionalmaterials immobilized thereon are hardly to say a convenient method. Onthe other hand, a method using the functional materials immobilized onbead(s) has the following advantages.

In comparison with a plate, immobilization on beads can be carried outat a relatively small space and beads can be handled in a sealed vessel.Since a surface of a plate having the functional material immobilizedthereon is exposed to air, it is necessary to take care to preventdeterioration or the like due to drying during storage in case thefunctional material having lower stability. However, beads can be storedby suspending in a solution and such troubles can be avoided. Moreover,a surface area of the functional material becomes larger by using beadsand therefore, in comparison with a plate, the higher gene transferefficiency can be obtained.

Immobilization of the functional materials can be carried out by theconventional method, for example, a target cell culture vessel is coatedwith the functional materials or the functional materials can beimmobilized, for example, on culture beads for culturing cells. The rawmaterial and kind of beads can be selected depending upon the intendeduse. For example, the bead may have a circular or spherical core as acentral portion and the surface of the core can be coated with ahydrophilic polymer. Examples of the raw material and the kind of thecore and the polymer are described in JP-A 8-501092. For example,biodegradable beads on which these functional materials are immobilizedmay be administered in a living body. Alternatively, an effective methodis to use a mixture of beads on which a molecule having a retrovirusbinding domain is immobilized and beads on which a molecule having atarget cell binding domain is immobilized.

When these functional materials are used without immobilization, forexample, a target cell culture vessel can be pre-treated with asubstance which prevents the functional materials from the adhesion tothe vessel, for example, bovine serum albumin (BSA). Thus, thefunctional materials can be used without non-specific adhesion to thevessel.

According to the present invention, gene transfer can be efficientlycarried out even in such a system that the functional material of thepresent invention is used without immobilization.

In addition, by using the reagent kit specifically designed for carryingout the method of the present invention as described hereinafter, genetransfer into cells can be very conveniently carried out.

As described above, transformant cells obtained according to the methodof the present invention can be grafted into a living body, thereby genetherapy can be carried out to express an exogenous gene in a livingbody.

For example, when hematopoietic stem cells are used as target cells,gene therapy can be carried out by the following procedures. First, amaterial containing the hematopoietic cells, for example, bone marrowtissue, peripheral blood, fetal umbilical cord blood or the like iscollected from a donor. The material can be used as such. However,usually, a monocyte fraction containing hematopoietic stem cells isprepared by density gradient centrifugation or the like. Alternatively,hematopoietic stem cells can be purified by utilizing markers on cellsurfaces such as CD34 and/or C-kit. The material containinghematopoietic cells can optionally be pre-stimulated with a suitablecell growth factor or the like and then, the cells are infected with arecombinant retroviral vector into which an intended gene has beeninserted according to the method of the present invention, inparticular, in the presence of the functional material having stem cellbinding activity. The transformant cells thus obtained can be graftedinto a recipient by, for example, intravenous administration. Therecipient is, preferably, an autologous donor but also includingallogeneic transplants, the latter especially where umbilical cord bloodcells are used for the graft.

Gene therapy using hematopoietic stem cells as target cells is tocompensate for a deficient or abnormal gene of a patient and examplesthereof include ADA deficiency and Gaucher's disease. In addition,sometimes, transduction of a drug resistant gene is carried out torelieve hematopoietic stem cell disorders due to chemotherapy used intreatment of cancers, leukemias and the like.

It has been known that hematopoietic stem cells express VLA-4 receptorand it is therefore possible to carry out gene transfer efficiently byusing the functional material having CS-1 cell adhesion region disclosedby the present invention. Further, as described above, molecules such asCD34 and C-kit are expressed on the surfaces of hematopoietic stem cellsand therefore the gene transfer efficiency can be improved by combiningantibodies against to these molecules or a stem cell factor which is aligand of C-kit with the functional material having retrovirus bindingdomain.

Moreover, as gene therapy of cancers, tumor vaccino-therapeutics havebeen studied, wherein cytokine genes are transferred into cancer cellsand, after depriving growth capability, the cells are returned to thepatient body to increase tumor immunity (Human Gene Therapy, Vol. 5, pp.153-164 (1994)). Such treatment can also be carried out effectively byapplying the method of the present invention with the functionalmaterial having high affinity to cancer cells.

Further, activities have been attempted to treat AIDS by gene therapy.In this case, it has been proposed to transfer a gene encoding a nucleicacid molecule which inhibits HIV replication or gene expression (e.g.,anti-sense nucleic acid, ribozyme, etc.) into T cells which is infectedwith HIV which causes AIDS (J. Virol., Vol. 69, pp. 4045-4052 (1995)).Gene transfer into T cells can be achieved by the method of the presentinvention with utilizing the functional material, for example, CD4antibody or the like which can bind to a molecule present on the surfaceof T cells.

Thus, as target cells for gene transfer, any cells can be used in so faras the functional material having target cell binding domain of thepresent invention is available or can be prepared.

Moreover, the method of the present invention is suitable for protocolsof clinical gene therapy because co-cultivation of target cells in thepresence of retrovirus producer cells is not required and the method ofthe present invention can be carried out in the absence ofhexadimethrine bromide whose use is clinically disadvantageous in humanbeing.

Further, as application of the present invention to art fields otherthan gene therapy, for example, transgenic vertebrate animals can besimply produced by using, as a target cells, embryoplastic stem cells,primordial germ cell, oocyte, oogonia, ova, spermatocyte, sperm and thelike.

That is, as one aspect, the present invention provides a method forcellular grafting comprising grafting the transformant cells obtained bythe method of the present invention into a vertebrate animal. Examplesof vertebrate animals to be grafted with transformant cells includemammals (e.g., mouse, rat, rabbit, goat, pig, horse, dog, monkey,chimpanzee, human being, etc.), birds (e.g., chicken, turkey, quail,duck, wild duck, etc.), reptiles (e.g, snake, alligator, tortoise,etc.), amphibian (e.g., frog, salamander, newt, etc.), fishes (e.g., dogmackerel, mackerel, bass, snapper, grouper, yellowtail, tuna, salmon,trout, carp, sweetfish, eel, flounder, shark, ray, sturgeon, etc.).

Thus, according to this aspect of the present invention, likesubstantially pure fibronectin, substantially pure fibronectin fragmentsor a mixture thereof, gene transfer with retroviruses can be carried outefficiently by the retrovirus binding domain and the target cell bindingdomain of the functional material to be used in the present invention.Then, the present invention can provide a technique for transferringgenetic materials into vertebrate cells without any limitation ofconventional techniques.

In a further aspect of the present invention, an effective amount of amaterial which has both retrovirus binding domain and target cellbinding domain on the same molecule and has functions equivalent tothose of substantially pure fibronectin, substantially pure fibronectinfragments or a mixture thereof is used as the functional material.

Such a functional material is a material which can perform gene transferwith the same efficiency as that of fibronectin, a fibronectin fragmentor a mixture thereof. Typically, it is the functional material havingthe above novel retrovirus binding domain and target cell binding domainof the present invention on the same molecule. In case of using thesematerials, it is considered that retroviruses as well as target cellsbind to at least one functional material.

Examples of the functional material having a retrovirus binding domainand a target binding domain on the same molecule include polypeptidesrepresented by SEQ. ID Nos. 21 and 22 of the Sequence Listing(hereinafter referred to as CHV-181 and CHV-179, respectively).

These peptides include type III similar sequences (III-12, III-13 andIII-14) contained in H-271. In CHV-181, III-12 and III-13 sequences, andin CHV-179, III-13 and III-14 sequences are added to the C-terminal ofthe cell adhesion polypeptide (Pro¹²³⁹-Ser¹⁵¹⁵) of fibronectin viamethionine. A plasmid for expressing the polypeptide CHV-181 can beconstructed, for example, by the following procedures.

First, the plasmid pHD101 containing a DNA fragment encoding the heparinbinding polypeptide (H-271) of fibronectin is prepared in Escherichiacoli HB101/pHD101 (FERM BP-2264). A HindIII site is introduced in aregion encoding the C-terminal of the III-13 sequence on this plasmid bysite-directed mutagenesis, followed by digestion with NcoI and HindIIIto obtain a DNA fragment encoding III-12 and III-13 sequence. On theother hand, the plasmid vector pINIII-ompA₁ is digested with HindIII andSalI to obtain a DNA fragment encoding a lipoprotein terminator region.

Next, the plasmid pTF7021 containing a DNA fragment encoding the celladhesion polypeptide (C-279) of fibronectin is prepared from Escherichiacoli JM109/pTF7021 (FERM BP-1941), and a NcoI site is introducedimmediately before termination codon of C-279 on the plasmid bysite-directed mutagenesis to obtain the plasmid pTF7520. This plasmid isdigested with NcoI and SalI, followed by mixing with the DNA fragmentencoding the III-12 and III-13 sequence and the DNA fragment encoding alipoprotein terminator region to ligate them to obtain the plasmidpCHV181 for expressing the polypeptide CHV-181. The nucleotide sequenceof a region encoding the polypeptide CHV-181 on the plasmid pCHV181 isshown in SEQ. ID No. 27 of the Sequence Listing.

A plasmid for expressing the polypeptide CHV-179 can be constructed, forexample, by the following procedures.

First, a NcoI site is introduced in a region encoding the N-terminal ofthe III-13 sequence on the plasmid pHD101 by site-directed mutagenesis,followed by digestion with NcoI and HindIII to obtain a DNA fragmentencoding the III-13 and III-14 sequence. This is mixed with a DNAfragment encoding the above lipoprotein terminator region and the NcoIand SalI-digested plasmid pTF7520 to ligate them to obtain the plasmidpCHV179 for expressing the polypeptide CHV-179.

CHV-181 and CHV-179 can be obtained by culturing E. coli transformedwith the above plasmids, respectively, then purifying from the resultingculture.

These functional materials can be used by immobilized on, for example,beads as described above or without immobilization.

In another aspect, the present invention provides a culture medium oftarget cells to be used for gene transfer into the target cells withretroviruses which comprises (1) the above-described mixture of aneffective amount of the functional material having retrovirus bindingdomain and an effective amount of another functional material having thetarget cell binding domain or (2) an effective amount of the functionalmaterial having the above described novel retrovirus binding domain andtarget cell binding domain on the same molecule. The functional materialmay be immobilized or may be used without immobilization.

Other ingredients of the culture medium of the present invention are notspecifically limited in so far as they can be used in culture of targetcells and commercially available culture mediums for culturing cells canbe used. The culture medium of the present invention can also containserum, a cell growth factor necessary for growth of target cells, anantibiotic for preventing contamination of microorganisms and the like.For example, in case of NIH/3T3 cell, Dulbecco's modified Eagle's medium(DMEM, JRH Bioscience) containing 10% bovine fetal serum (Gibco), 50units/ml of penicillin and 50 μg/ml of streptomycin (both Gibco) can beused as the culture medium.

In further aspect, the present invention provides a method forlocalization of a retrovirus which comprises incubating a culture mediumcontaining the retrovirus contacted with (1) the above-described mixtureof a molecule containing the retrovirus binding domain and anothermolecule containing the target cell binding domain, (2) theabove-described functional material having the novel retrovirus bindingdomain of the present invention and a target cell binding domain on thesame molecule, or (3) the above-described functional material having theretrovirus binding domain.

As described above, the functional material may be immobilized or may beused without immobilization. Incubation can be carried out according toa conventional method, for example, at 37° C. under the conditions ofCO₂ concentration of 5% and humidity of 99.5%. These conditions can besuitably adjusted depending on particular target cells to be used andthe culture period can also be changed according to particular cells andpurposes.

By using the method of the present invention, viral particles can belocalized in various constructs which deliver viruses into target cells.

In another aspect of the present invention, there is provided a kit forusing retrovirus-mediated gene transfer into target cells. The kitcomprises:

(a) an effective amount of (1) a mixture of the molecule having theabove described retrovirus binding domain and another molecule havingthe target cell binding domain, or (2) the functional material havingthe novel retrovirus binding domain of the present invention and thetarget cell binding domain on the same molecule;

(b) an artificial substrate for incubating the retrovirus contacted withthe target cells; and

(c) a target cell growth factor for pre-stimulating the target cells.The functional material (a) may be immobilized or non-immobilized. Thiskit may further comprise a recombinant retroviral vector, necessarybuffers and the like.

As the artificial substrate, there can be used plates for culturingcells, petri dishes, flasks and the like. They may be made ofpolystyrene.

In case that target cells are cells in G₀ phase, infection with aretrovirus does not occur and therefore, preferably, cells arepre-stimulated to lead cells to the cell cycle. For this purpose, targetcells are cultured in the presence of a suitable cell growth factorprior to infection with a retrovirus. For example, in case of genetransfer into bone marrow cells and hematopoietic stem cells, a targetcell growth factor such as Interleukin-6 or a stem cell factor can beused.

Respective constituent members of the kit can be prepared in the form offreeze dried products, granules, tablets in addition to aqueoussolutions according to per se known methods.

By using the kit of the present invention, for example, a transformedviable target cell culture can be obtained and retrovirus-mediatedtransduction into target cells can be simply carried out.

The present invention also includes a method for gene transfer intotarget cells with retrovirus wherein the functional material selectedfrom the group consisting of substantially pure fibronectin,substantially pure fibronectin fragments and a mixture thereof, or apolymer thereof which is immobilized on beads or not immobilized isused.

The present invention includes the above described CH2-826 and itsfunctional equivalents. In addition, the present invention provides agene encoding CH2-826. One example thereof is a gene represented by SEQ.ID No. 20 of the Sequence Listing. The present invention also includesfunctional equivalents of the gene.

Further, the present invention provides the above described CHV-181 andincludes its functional equivalents. In addition, the present inventionprovides a gene encoding CHV-181. One example of the gene is thatrepresented by SEQ. ID No. 27 of the Sequence Listing. The presentinvention also includes functional equivalents of the gene.

The present invention also provides a polymer containing a polymer ofthe retrovirus binding domain and/or a polymer of the target cellbinding domain. Specific examples of the polymer are a polymer of afibroblast growth factor and a polymer of a polypeptide having aninsulin binding domain derived from type V collagen.

As discussed hereinafter, although the present invention is not limitedby any theory, it is believed that gene transfer into cells with aretrovirus, i.e., transformation is enhanced by binding the retrovirusand the target cell to respective functional domains.

As such a functional material which binds to a retrovirus and thus isuseful in the present invention, there are substantially purefibronectin, substantially pure fibronectin fragments or a mixturethereof. The present inventors have found that the above-describedfunctional materials of the present invention having functionssubstantially the same as those of substantially pure fironectin and thelike improve the gene transfer efficiency, i.e., the transformationefficiency of target cells with a retrovirus.

The fragments of fibronectin described herein may be of natural orsynthetic origin and can be prepared in substantial purity fromnaturally occurring materials, for example as previously described byRuoslahti et al. (1981) J. Biol. Chem. 256:7277; Patel and Lodish (1986)J. Cell. Biol., 102:449; and Bernardi et al. (1987) J. Cell. Biol.105:489. In this regard, reference herein to substantially purefibronectin or a fibronectin fragment is intended to mean that they areessentially free from other proteins with which fibronectin naturallyoccurs.

The substantially pure fibronectin or fibronectin fragment describedherein can also be produced by genetic engineering techniques, forexample, as generally described in U.S. Pat. No. 5,198,423. Inparticular, the recombinant fragments identified in the Examples belowas H-271, H-296, CH-271 (SEQ ID NO 23) and CH-296 (SEQ ID NO 24), andmethods for obtaining them, are described in detail in this patent. TheC-274 fragment used in the Examples below was obtained as described inU.S. Pat. No. 5,102,988. These fragments or fragments from which theycan be routinely derived are available by culturing E. coli depositedwith NIBH of 1-1-3, Higashi, Tsukuba-sh, Ibaraki-ken, Japan underBudapest Treat with the accession numbers of FERM P-10721 (H-296) (thedate of original deposit: May 12, 1989), FERM BP-2799 (C-277 bound toH-271 via methionine) (the date of original deposit: May 12, 1989), FERMBP-2800 (C-277 bound to H-296 via methionine) (the date of originaldeposit: May 12, 1989) and FERM BP-2264 (H-271) (the date of originaldeposit: Jan. 30, 1989), as also described in U.S. Pat. No. 5,198,423.

In addition, useful information as to fibronectin fragments utilizableherein or as to starting materials for such fragments may be found inKimizuka et al., J. Biochem. 110, 284-291 (1991), which reports furtheras to the above-described recombinant fragments; in EMBO J., 4,1755-1759 (1985), which reports the structure of the human fibronectingene; and in Biochemistry, 25, 4936-4941 (1986), which reports on theHeparin-II binding domain of human fibronectin. Fibronectin fragmentswhich contain both the CS-l cell adhesion domain and the Heparin-IIbinding domain have been found to significantly enhance the efficiencyof gene transfer into hematopoietic cells in work thus far.

It will thus be understood that the fibronectin-related polypeptidesdescribed herein will provide an amino acid sequence having thecell-binding activity of the CS-l cell adhesion domain of fibronectin aswell as an amino acid sequence of the Heparin-II binding domain offibronectin which binds the virus.

The viral-binding polypeptide utilized to enhance transduction byretroviral vectors as disclosed in WO 95/26200 will comprise (i) a firstamino acid sequence which corresponds to the Ala¹⁶⁹⁰-Thr¹⁹⁶⁰ of theHeparin-II binding domain of human fibronectin, which is represented bythe formula (SEQ ID NO 1):

Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr Ser LeuSer Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg ValThr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp SerSer Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser ValTyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr ThrLeu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu ThrThr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln ValAsp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Sys Pro AspVal Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile TyrLeu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala SerThr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn SerLeu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile LysTyr Glu Sys Pro Gly Sev Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro GlyVal Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile TyrVal Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys LysThr;or a sufficiently similar amino acid sequence thereto to exhibit theability to bind the retrovirus;and (ii) a second amino acid sequence (CS-1) which corresponds to oneportion of the IIICS binding domain of human fibronectin; which isrepresented by the formula (SEQ. ID No. 2):

-   Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro    Glu Ile Leu Asp Val Pro Ser Thr;-   or a sufficiently similar amino acid sequence thereto to exhibit the    ability to bind hematopoietic cells such as primitive progenitor    and/or long term repopulating (stem) cells.

The retrovirus binding activity of a polypeptide represented by theabove SEQ. ID No. 1 (H-271) shows a concentration dependence and, asindicated in Example 8 below, it shows substantially the same activityas that of CH-271 at high concentrations. That is, a retrovirus andtarget cells bind to at least one molecule of H-271 for the first timein the presence of a high concentration of H-271.

The strong virus binding to the virus binding domain of the functionalmaterial of the present invention can be used for constructing deliverysystems for virus-mediated therapy across a broad range of cell types.For this purpose, a polypeptide containing the retrovirus binding domainof the functional material of the present invention can be coupled toany material containing a cell binding domain which gives this constructspecificity for the target cells, or can be co-localized with a materialcontaining its cell binding domain. That is, the virus bindingpolypeptide may be covalently coupled to the cell binding material orthey may be different molecules.

This approach will circumvent the prior necessity of constructingspecific retrovirus cell lines for each target cell and facilitateselection of the functional material having the most suitable targetcell binding domain according to a particular kind of target cells.Therefore, by using the functional material of the present invention,transduction specific for target cells to be used can be readily carriedout and, in particular, the method of the present invention wherein amixture of the functional material having retrovirus binding domain andthe functional material having target cell binding domain is especiallyuseful for transfer the required gene into the intended target cell. Inaddition, the novel functional material provided by the presentinvention is especially useful for the method for improving the genetransfer efficiency into target cells with retroviruses and relatedtechniques.

The following Examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

EXAMPLE 1

(1) Preparation of Virus Supernatant

GP+E-86 producer cells (ATCC CRL-9642) containing retroviral plasmidPM5neo vector containing a neomycin resistant gene (Exp. Hematol., 23,630-638 (1995)) were cultured in Dulbecco's modified Eagle medium (DMEM,JRH Bioscience) containing 10% fetal calf serum (FCS, Gibco) and 50units/ml of penicillin and 50 μg/ml of streptomycin (both Gibco). AllDMEM used hereinafter contained 50 units/ml of penicillin and 50 μg/mlof streptomycin. PM5neo virus containing supernatant was collected byadding 4 ml of DMEM containing 10% FCS to semi-confluent plates toculture overnight. Harvested medium was filtered through 0.45 micronfilters (Millipore) to obtain virus supernatant which was stored at −80°C. until used.

Separately, regarding retrovirus plasmid, TKNEO vector (Blood, 78,310-317 (1991)), TKNeo virus supernatant was prepared according to thesame procedures as those described above by using GP+envAm-12 cells(ATCC CRL-9641).

(2) Determination of Virus Titer of Supernatant

The virus titer of the supernatant was determined by using NIH/3T3 cellsaccording to the standard method (J. Virol., 62, pp. 1120-1124 (1988)).Namely, DMEM and 2,000 cells/well of NIH/3T3 cells were added to a6-well tissue culture plate. After cultivation overnight, the seriallydiluted virus supernatant was added to each well together withhexadimethrine bromide (polybrene manufactured by Aldrich) at the finalconcentration of 7.5 μg/ml. This was incubated at 37° C. for 24 hoursand then the medium was replaced by that containing G418 (Gibco) at thefinal concentration of 0.75 mg/ml. The plate was further incubated. G418resistant (G418^(r)) colonies which grew after 10 to 12 days werestained with crystal violet to record their count. The number ofinfectious particles per 1 ml of the supernatant (cfu/ml) was calculatedby multiplying the number of colonies per well by the dilution rate andit was used as the titer of the supernatant to determine the amount ofthe virus supernatant to be added in the subsequent experiments.

EXAMPLE 2

(1) Preparation of Polypeptide Derived from Fibronectin

The polypeptide derived from human fibronectin, H-271 (amino acidsequence is shown in SEQ. ID No. 1 of the Sequence Listing) was preparedfrom E. coli containing the recombinant plasmid containing DNA encodingthe polypeptide, pHD101, i.e., Escherichia coli HB101/pHD101 (FERMBP-2264) according to the method disclosed in U.S. Pat. No. 5,198,423.

The polypeptide, CH-271 (amino acid sequence is shown in SEQ. ID No. 23of the Sequence Listing) was prepared as follows. Namely, Escherichiacoli HB101/pCH101 (FERM BP-2799) was cultured according to the methoddescribed in the above patent and CH-271 was obtained from the culture.

And, the polypeptide, CH-296 (amino acid sequence is shown SEQ. ID No.24) was prepared as follows. Namely, Escherichia coli HB101/pCH102 (FERMBP-2800) was cultured according to the method described in the abovepatent and CH-296 was obtained from the culture.

The polypeptide, C-274 (amino acid sequence is shown in SEQ. ID No. 25of the Sequence Listing) was prepared as follows. Namely, Escherichiacoli JM109/pTF7221 (FERM BP-1915) was cultured according to the methoddescribed in U.S. Pat. No. 5,102,988 and C-274 was obtained from theculture.

Further, the polypeptide, C277-CS1 (amino acid sequence is shown in SEQ.ID No. 29 of the Sequence Listing) was prepared as follows. Namely,Escherichia coli HB101/pCS25 which was disclosed in JP-A 3-284700 underFERM P-11339 and was deposited with the above NIBH of 1-1-3, Higashi,Tsukubashi, Ibaraki-ken under Budapest Treaty with the accession numberFERM BP-5723 (date of original deposit: Mar. 5, 1990) was culturedaccording to the method described in the above patent and C277-CS1 wasobtained from the culture.

(2) Preparation of C-FGF•A

The polypeptide, C-FGF•A (amino acid sequence is shown in SEQ. ID No. 4of the Sequence Listing) was prepared as follows. Namely, E. colicontaining the recombinant plasmid containing DNA encoding the abovepolypeptide, pYMH-CF•A, i.e., Escherichia coli JM109/pYMH-CF•A (FERMBP-5278) was cultured in 5 ml of LB broth containing 100 μg/ml ofampicillin at 37° C. for 8 hours. This pre-culture broth was inoculatedinto 500 ml of LB broth containing 100 μg/ml of ampicillin and 1 mM ofIPTG (isopropyl-β-D-thiogalactopyranoside) and cultivated at 37° C.overnight. The microbial cells were harvested, suspended in 10 ml of PBS(phosphate buffered saline) containing 1 mM PMSF (phenylmethanesulfoniumfluoride) and 0.05% of Nonidet P-40 and sonicated to disrupt the cells.The mixture was centrifuged to obtain a supernatant. To absorbance 4,000at 260 nm of this supernatant was added 1 ml of 5% polyethylene imineand the mixture was centrifuged to obtain a supernatant. The supernatantwas applied to a HiTrap-Heparin column (Pharmacia) equilibrated withPBS. After washing the non-absorbed fraction with PBS, the absorbedfraction was eluted with PBS containing NaCl gradient of from 0.5 M to 2M. The eluate was analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) which showed the presence of two fractions containing 47 kdpolypeptide. One fraction of them which was eluted at the higher NaClconcentration was collected and applied to a Superose 6 column(Pharmacia) equilibrated with PBS containing 1.5 M NaCl. The eluate wasanalyzed by SDS-PAGE and a fraction containing about 47 kd polypeptidewas collected to obtain the purified C-FGF•A which was used in thesubsequent steps.

(3) Preparation of C-FGF-CS1

First, a plasmid was constructed for expressing the polypeptide,C-FCF-CS1 (amino acid sequence is shown in SEQ. ID No. 5 of the SequenceListing) in Escherichia coli as a host.

Escherichia coli HB101/pCH102 (FERM BP-2800) was cultured and theplasmid pCH102 was prepared by alkali-SDS method from the resultingmicrobial cells. PCR was carried out using this plasmid as a template aswell as primer M4 (Takara Shuzo Co., Ltd.) and primer CS1-S, nucleotidesequence of which is shown in SEQ. ID No. 9 in the Sequence Listing andan amplified DNA fragment in the reaction solution was recovered withethanol precipitation. The resultant DNA fragment was digested with NheIand SalI (both Takara Shuzo Co., Ltd.), followed by agarose gelelectrophoresis to recover about 970 bp DNA fragment from the gel.

Escherichia coli JM109/pYMH-CF•A (FERM BP-5278) was then cultured andthe plasmid pYMH-CF•A was prepared by an alkali-SDS method from theresulting microbial cells. PCR reaction was carried out using thisplasmid as a template as well as primer CF, nucleotide sequence of whichis shown in SEQ. ID No. 10, and primer FNR, nucleotide sequence of whichis shown in SEQ. ID No. 11 of the Sequence Listing, and an amplified DNAfragment in the reaction solution was recovered with ethanolprecipitation. The resultant DNA fragment was digested with Eco52I(Takara Shuzo Co., Ltd.) and NheI, followed by agarose gelelectrophoresis to recover about 320 bp DNA fragment from the gel.

About 4.1 kb DNA fragment isolated by digesting the plasmid pYMH-CF•Awith Eco52I and SalI and subjecting to agarose gel electrophoresis wasmixed with the above 970 bp DNA fragment and about 320 bp DNA fragmentto ligate them to obtain a recombinant plasmid which was inserted intoE. coli JM109. A plasmid was prepared from the resulting transformantand that containing each one molecule of the above three DNA fragmentswas selected and named plasmid pCFS100. E. coli JM109 transformed withthe plasmid pCFS100 was named Escherichia coli JM109/pCRS100. Theplasmid pCFS100 has a CS-1 cell adhesion region derived from fibronectinat the C-terminal of C-FGF•A and encodes the polypeptide, C-FGF-CS1,wherein second lysine from the C-terminal of FGF was substituted withalanine.

The polypeptide, C-FGF-CS1 was prepared as follows. Namely, the above E.coli JM109/pCFS100 was cultured in 5 ml of LB broth containing 100 μg/mlof ampicillin at 37° C. for 8 hours. This pre-cultured broth wasinoculated into 500 ml of LB broth containing 100 μg/ml of ampicillinand 1 mM IPTG and cultured overnight at 37° C. to collect the microbialcells. The resulting microbial cells were suspended in 10 ml of PBS(phosphate buffered saline) containing 0.5M NaCl, 1 mM PMSF and 0.05%Nonidet P-40, and the microbial cells were sonicated to disrupt andcentrifuged to obtain a supernatant. This supernatant was subjected toHiTrap-Heparin column pre-equilibrated with PBS containing 0.5 M NaCl,the non-adsorbed fractions were washed with PBS containing 0.5 mM NaCland the adsorbed fraction was eluted with PBS having a concentrationgradient of 0.5 M to 2 M NaCl. The eluate was analyzed bySDS-polyacrylamide gel electrophoresis and fractions containing about 50kd polypeptide were collected to obtain purified C-FGF-CS1 which wasused in the subsequent steps.

Amino acid sequence of from N-terminal to the fifth amino acid ofpurified C-FGF-CS1 thus obtained was investigated and found to beconsistent with that shown in SEQ. ID No. 5 of the Sequence Listing. Inaddition, molecular weight of purified C-FGF-CS1 measured bymasspectroscopy was consistent with that expected from the above aminoacid sequence.

(4) Preparation of C277-ColV

The polypeptide, C277-ColV (amino acid sequence is shown in SEQ. ID No.6 of the Sequence Listing) was purified as follows. Namely, E. colicontaining the recombinant plasmid containing DNA encoding the abovepolypeptide, pTF7520ColV, i.e., Escherichia coli JM109/pTF7520 ColV(FERM BP-5277) was cultured in 5 ml of LB broth containing 100 μg/ml ofampicillin at 37° C. for 6.5 hours. This pre-culture broth wasinoculated into 500 ml of LB broth containing 100 μg/ml ampicillin andcultivated at 37° C. When the absorbance at 660 nm reached 0.6, IPTG wasadded to the broth to make up to 1 mM of the final concentration and thebroth was cultured overnight to harvest microbial cells. The microbialcells obtained were suspended in 10 ml of PBS containing 1 mM of EDTA,0.05% of Nonidet P-40 and 2 mM of PMSF, and sonicated for 10 minutes todisrupt the cells. The cell disruption solution was centrifuged and theresultant supernatant was applied to a Resource Q column (Pharmacia) toobtain a non-adsorbed fraction containing the desired polypeptide. Thefraction was applied to HiTrap-Heparin column equilibrated with PBS.After washing the non-adsorbed fraction with PBS, the adsorbed fractionwas eluted with PBS having NaCl gradient of from 0 M to 0.5 M NaCl. Theeluate was analyzed by SDS-PAGE and the fractions containing 48 kdpolypeptide were collected to obtain the purified C277-ColV which wasused in the subsequent steps.

(5) Preparation of ColV

First, a plasmid was constructed for expressing the polypeptide, ColV(amino acid sequence is shown in SEQ. ID No. 6 of the Sequence Listing)in Escherichia coli as a host.

Escherichia coli HB101/pTF7520ColV (FERM BP-5277) was cultivated and theplasmid pTF7520ColV was prepared by alkali-SDS method from the resultingmicrobial cells. This plasmid was digested with NcoI and BamHI (bothTakara Shuzo Co., Ltd.), followed by agarose gel electrophoresis torecover about 0.58 kb DNA fragment from the gel. This was mixed with theplasmid vector pET8C (Novagen) predigested with NcoI and BamHI to ligatethem. The resultant recombinant plasmid was introduced into E. coli BL21to obtain a transformant, from which plasmids were prepared, a plasmidcontaining only one molecule of the above about 0.58 kb DNA fragment wasselected and named pETColV.

E. coli BL21 transformed with the above plasmid pETColV, that is,Escherichia coli BL-21/pETColV was cultured overnight in 10 ml of LBbroth containing 50 μg/ml of ampicillin at 37° C. 0.2 ml of thispre-culture solution was inoculated into 100 ml of L-broth containing 50μg/ml of ampicillin, followed by cultivating at 37° C. When theabsorbance at 600 nm reached 0.4, IPTG was added thereto at the finalconcentration of 1 mM, followed by cultivating overnight to collect themicrobial cells. The resulting microbial cells were suspended in 5 ml ofPBS (phosphate buffered saline) containing 1 mM of EDTA, 0.05% ofNonidet P-40, 10 μg/ml of aprotinin, 10 μg/ml of leupeptin and 2 mM ofPMSF, the cells were sonicated to disrupt, followed by centrifugation toobtain a supernatant. This supernatant was subjected to a HiTrap-Heparincolumn equilibrated with PBS, the non-adsorbed fractions were washedwith PBS and the adsorbed fraction was eluted with PBS containing 0.5MNaCl. The eluate was analyzed by SDS-polyacrylamide gel electrophoresisand almost homogenious about 18 kd polypeptide was confirmed. PurifiedColV thus obtained was used in the subsequent steps.

(6) Preparation of H2-547

A plasmid for expressing the polypeptide, H2-547 (amino acid sequence isshown in SEQ. ID No. 13 of the Sequence Listing) was constructed asfollows. Escherichia coli HB101/pCH101 (FERM BP-2800) was cultivated andthe plasmid pCH102 was prepared from the resultant cells usingalkali-SDS method. PCR was carried out using this plasmid as a templateas well as primer 12S, the nucleotide sequence of which is shown in SEQ.ID No. 15 of the Sequence Listing, and primer 14A, the nucleotidesequence of which is shown in SEQ. ID No. 16 of the Sequence Listing,followed by agarose gel electrophoresis to recover an about 0.8 kb DNAfragment encoding a heparin binding polypeptide of fibronectin from thegel. The resulting DNA fragment was digested with NcoI and BamHI (bothTakara Shuzo Co., Ltd.) and mixed with NcoI-BamHI digested pTV118N(Takara Shuzo Co., Ltd.) to ligate them, which was inserted into E. coliJM109. Plasmids were prepared from the resulting transformant and aplasmid containing the above DNA fragment was selected and maned plasmidpRH1.

The plasmid vector, pINIII-ompA₁ (The EMBO Journal, 3, 3437-2442 (1984))was digested with BamHI and HincII (Takara Shuzo Co., Ltd.) to recoveran about 0.9 kb DNA fragment containing a lipoprotein terminator region.This was mixed with BamHI-HincII digested plasmid pRH1 to ligate them toobtain the plasmid pRH1-T containing lac promoter, DNA fragment encodinga heparin binding polypeptide and lipoprotein terminator in this order.

An about 3.1 kb DNA fragment obtained by digesting the plasmid pRH1-Twith NheI and ScaI (both Takara Shuzo Co., Ltd.) and an about 2.5 kb DNAfragment obtained by digesting the plasmid pRH1-T with Spe I (TakaraShuzo Co., Ltd.) and ScaI were prepared, respectively, and these twofragments were ligated to obtain the plasmid pRH2-T containing lacpromoter, open reading frame encoding a polypeptide wherein two heparinbinding polypeptides are connected in tandem, and lipoprotein terminatorin this order. A nucleotide sequence of the above open reading frame isshown SEQ. ID No. 17 of the Sequence Listing.

The polypeptide, H2-547 was prepared as follows. Four 500 ml Erlenmeyerflasks, equipped with a baffle, containing 120 ml of LB broth containing100 μg/ml of ampicillin were prepared, these were inoculated with E.coli HB101 transformed with the above plasmid pRH2-T, that is,Escherichia coli HB101/pRH2-T to culture overnight at 37° C. Themicrobial cells were collected from the culture by centrifugation,suspended in a 40 ml disruption buffer (50 mM tris-HCl, 1 mM EDTA, 150mM NaCl, 1 mM DTT, 1 mM PMSF, pH 7.5) and the microbial cells weresonicated to disrupt. The supernatant obtained by centrifugation wassubjected to High trap Heparin column (Pharmacia) equilibrated with apurification buffer (50 mM tris-HCl, pH 7.5). The non-adsorbed fractionsin the column were washed with the same buffer, followed by elution witha purification buffer having the concentration gradient of 0 to 1 MNaCl. The eluate was analyzed with SDS-polyacrylamide gelelectrophoresis and the fractions containing a polypeptide having themolecular weight of about 60,000 were collected to obtain purifiedH2-547 preparation. The protein amount contained in the resultingpreparation was analyzed with BCA PROTEIN ASSAY REAGENT (Pierce) usingbovine serum albumin as a standard, indicating that about 10 mg ofH2-547 was obtained.

Amino acid sequence of from the N-terminal to the fifth residue ofpurified H2-547 thus obtained was investigated and found to beconsistent with amino acid sequence of H2-547 expected from nucleotidesequence shown in SEQ ID NO 17 of the Sequence Listing minus methionineat the N-terminal (sequence thereof is shown in SEQ. ID No. 13 of theSequence Listing). The molecular weight of purified H2-547 measured bymasspectroscopy was consistent with that expected from amino acidsequence shown in SEQ. ID No. 13 of the Sequence Listing.

(7) Preparation of CH2-826

A plasmid for expressing the polypeptide, CH2-826 (amino acid sequenceis shown in SEQ. ID No. 14 of the Sequence Listing) was constructed asfollows. PCR was carried out using the above plasmid pCH102 as atemplate as well as primer CLS, the nucleotide sequence of which isshown in SEQ. ID No. 18 of the Sequence Listing, and primer CLA, thenucleotide sequence of which is shown in SEQ. ID No. 19 of the SequenceListing, followed by agarose gel electrophoresis to recover an about 0.8kb DNA fragment encoding the cell adhesion polypeptide of fibronectin.The resulting DNA fragment was digested with NcoI and BglII (both TakaraShuzo Co., Ltd.) and mixed with NcoI-BamHI digested pTV118N to ligatethem, which was inserted into E. coli JM109. Plasmids were prepared fromthe resulting transformant and a plasmid containing the above DNAfragment was selected and named plasmid pRC1. An about 2.5 kb DNAfragment obtained by digesting this plasmid pRC1 with SpeI and ScaI andan about 3.9 kb DNA fragment obtained by digesting the above plasmidpRH2-T with NheI and ScaI were mixed to ligate them to obtain theplasmid pRCH2-T encoding a polypeptide wherein two heparin bindingpolypeptides are tandemly connected to the C-terminal of the celladhesion polypeptide. A nucleotide sequence of open reading frame on theplasmid pRCH2-T encoding this polypeptide is shown in SEQ. ID No. 20 ofthe Sequence Listing.

The polypeptide, CH2-826 was prepared according to the same method asthat used for the polypeptide H2-547 described in Example 2 (6). Thefractions containing a polypeptide having the molecular weight of about90,000 were collected from the eluate of High trap Heparin column toobtain purified CH2-826.

(8) Preparation of H2S-537

A plasmid for expressing the polypeptide, H2S-537 (amino acid sequenceis shown in SEQ. ID No. 30 of the Sequence Listing) was constructed asfollows. PCR was carried out using the above plasmid pCH102 as atemplate as well as primer CS1S, the nucleotide sequence of which isshown in SEQ. ID No. 31 of the Sequence Listing, and primer CS1A, thenucleotide sequence of which is shown in SEQ. ID No. 32 of the SequenceListing, followed by agarose gel electrophoresis to recover an about 0.1kb DNA fragment encoding the cell adhesion polypeptide of fibronectin.The resulting DNA fragment was digested with NcoI and BamHI (both TakaraShuzo Co., Ltd.) and mixed with NcoI-BamHI digested pTV118N to ligatethem, which was inserted into E. coli JM109. Plasmids were prepared fromthe resulting transformant and a plasmid containing the above DNAfragment was selected and named plasmid pRS1.

The plasmid vector, pINIII-ompA₁ was digested with BamHI and HincII torecover an about 0.9 kb DNA fragment containing a lipoprotein terminatorregion. This was mixed with BamHI-HincII digested plasmid pRS1 to ligatethem to obtain the plasmid pRS1-T containing lac promoter, DNA fragmentencoding CS-1 region polypeptide and lipoprotein terminator in thisorder.

An about 2.4 kb DNA fragment obtained by digesting this plasmid pRS1-Twith NheI and ScaI and an about 3.3 kb DNA fragment obtained bydigesting the above plasmid pRH2-T with SpeI, ScaI and PstI (TakaraShuzo Co., Ltd.) were prepared. They were ligated to obtain the plasmidpRH2S-T containing lac promoter, open reading frame encoding apolypeptide having such structure in which two heparin bindingpolypeptides are tandemly connected and CS-1 region is further coupledto the C-terminal thereof, and lipoportein terminator in this order. Anucleotide sequence of the above open reading frame is shown in SEQ. IDNo. 32 of the Sequence Listing.

The polypeptide, H2S-573 was prepared according to the same method asthat used for the polypeptide H2-547 described in Example 2 (6). Thefractions containing a polypeptide having the molecular weight of about60,000 were collected from the eluate of High trap Heparin column toobtain purified H2S-573.

(9) Immobilization of Functional Material on Plate

For using a plate on which the functional material was immobilized inthe experiment for infection of cells with a retrovirus (6-well tissueculture plate, Falcon), immobilization was carried out according to thefollowing procedures. Namely, a solution of each functional materialdescribed the above Examples dissolved in PBS at a suitableconcentration was added to a plate at an amount of 2 ml per well (bottomarea 9.6 cm²) and the plate was incubated under UV light at roomtemperature for one hour without its cover and then for additional onehour with its cover. Then, the polypeptide solution was exchanged to 2ml of PBS containing 2% bovine serum albumin (BSA, Boehringer Mannheim)and incubated at room temperature for 30 minutes. The plate was washedwith PBS containing 25 mM of HEPES. A control plate coated on which BSAwas immobilized was prepared according to the same manner as indicatedabove except that the incubation with the polypeptide solution was notcarried out.

In gene transfer (virus infection) experiment in Examples below, theabove 6-well tissue culture plate was used unless otherwise indicated.When the concentration of the functional material used forimmobilization on the plate is indicated, an polypeptide amount per unitbottom area of a well is described using as unit of pmol/cm² (andμg/cm²). For example, when immobilization is carried out by using 2 mlof 48 μg/ml of H-271 solution on the above plate (bottom area 9.6 cm²),the description is “immobilization was carried out with 333 pmol/cm² (10μg/cm²) of H-271”. And, CH-296 immobilized plate to be used forculturing non-adherent cells (TF-1, HL-60) after transduction was thatprepared by immobilization of 48 pmol/cm² (3 μg/cm²) of a CH-296solution according to the above procedures. In the subsequent Examples,virus infection of target cells was always carried out in a mediumwithout polybrene. When an amount of virus, cell, medium and the likeare indicated, the amount per well is described unless otherwiseindicated.

EXAMPLE 3

(1) Gene Transfer Using Mixture of Functional Materials

The following experiment was carried out to investigate the effect onthe gene transfer in case of immobilization of a mixture of a cellbinding material and a retrovirus binding material on a plate. First,each polypeptide was immobilized on a plate by using 32 pmol/cm² (1.5μg/cm²) of C-FGF•A, a mixture 32 pmol/cm² (1 μg/cm²) of C-274 and 32pmol/cm² (0.5 μg/cm²) of FGF or 32 pmol/cm² (0.5 μg/cm²) of FGF (BectonDickinson) according to the same manner as described in Example 2 (9).After pre-incubating 2 ml of a virus supernatant containing 1,000 cfu ofPM5neo virus in respective plates and a control plate coated with BSA at37° C. for 30 minutes, the plates were thoroughly washed with PBS. Toeach of these plates was added 2 ml of DMEM medium containing 2,000NIH/3T3 cells and incubated at 37° C. for 2 hours in the absence ofpolybrene. Non adhered cells were collected by decantation and cellsadhered to the plate were collected by trypsin treatment to detach themfrom the plate. The cells were combined. The resultant cell suspensionwas divided into two halves. One half portion was cultured in DMEM andthe other portion was cultured in DMEM containing G418 at a finalconcentration of 0.75 mg/ml. Both portions were incubated at 37° C. for10 days and the colonies appeared were counted. By taking the ratio ofthe number of G418 resistant (G418^(r)) colonies relative to thatobtained in the medium without G418 as the gene transfer efficiency, theresults are shown in FIG. 1. In FIG. 1, the abscissa indicates thefunctional materials used and the ordinate indicates the gene transferefficiency.

As shown in FIG. 1, in case of 2 hour retrovirus infection, when themixture of C-274 and FGF was used, G418^(r) colonies were obtained atalmost the same gene transfer efficiency as that of C-FGF•A where thesetwo polypeptides were covalently coupled, though the gene transferefficiency obtain by using FGF alone was lower than that of C-FGF•A.

In order to investigate in detail, the effect of immobilization of C-274alone and FGF alone was compared with that of immobilization of amixture thereof. Namely, assessment was carried out except that platesprepared with 32 pmol/cm² (1 μg/cm²) of C-274, 32 pmol/cm² (0.5 μg/cm²)of FGF and a mixture of 32 pmol/cm² of C-274 and 32 pmol/cm² of FGF,respectively, according to the same manner as described in Example 2(9). The results are shown in FIG. 2. In FIG. 2, the abscissa indicatesthe functional materials used and the ordinate indicates the genetransfer efficiency.

As shown in FIG. 2, when using the plate on which immobilization wascarried out with the mixture of C-274 and FGF, the gene transferefficiency was higher than that using the plate on which only FGF wasimmobilized. And, no G418^(r) colonies appeared in the plate on whichimmobilization was carried out with C-274 which did not have anyretrovirus binding domain. This shows that, by combining FGF which hasthe retrovirus binding domain with C-274 which has the cell bindingdomain, the higher gene transfer efficiency can be obtained incomparison with that obtained by using FGF alone and that covalentcoupling of the polypeptides is not necessary required for elaboratingsuch effect of the combination of the polypeptides.

(2) Gene Transfer Using Mixture of Functional Materials

According to the same manner as described in Example 3 (1), assessmentwas carried out except that the polypeptide having retrovirus bindingdomain was replaced with ColV. In this experiment, the effect wasinvestigated by mixing C-274 and ColV in various molar ratios. Namely,according to the same manner as described in Example 2 (9),immobilization on plates was carried out by using 330 pmol/cm² (6μg/cm²) of ColV, a mixture of 330 pmol/cm² (10 μg/cm²) of C-274 and 330pmol/cm² of ColV (molar ratio of C-274:ColV=10:10), a mixture of 100pmol/cm² (3 μg/cm²) of C-274 and 330 pmol/cm² of ColV (3:10), a mixtureof 33 pmol/cm² (1 μg/cm²) of C-274 and 330 pmol/cm² of ColV (1:10), 330pmol/cm² (16 μg/cm²) of C277-ColV and 330 pmol/cm² (10 μg/cm²) of C-274,respectively. By using the plates thus prepared, the effect ofretrovirus infection was investigated according to the same manner asdescribed above. The results are shown in FIG. 3. In FIG. 3, theabscissa indicates the functional materials used and the ordinateindicates the gene transfer efficiency.

As shown in FIG. 3, in case of 2 hour infection, the infectionefficiency of ColV immobilized plate was less than ½ of that ofC277-ColV immobilized plate, while the infection efficiency of the plateon which immobilization was carried out with the mixture of ColV and its1/10 amount (as the molecular number) of C274 was the same as that ofC277-ColV immobilized plate. Then, the retrovirus infection enhancingactivity of C-274 was ascertained as observed in the case of FGF. Thiseffect was rather decreased in case that the amount of C-274 moleculesrelative to ColV molecules was increased. When a mixture containing thesame amounts of ColV and C-274 was coated, there was no substantialdifference between the mixture and ColV alone.

(3) Gene Transfer Using Mixture of Functional Materials

In order to investigate the effect on the gene transfer efficiency byimmobilization of a mixture of a material having cell binding domain anda material having retrovirus binding domain, the following experimentwas carried out. First, according to the same manner as described inExample 2 (9), immobilization of plates was carried out with 32 pmol/cm²(1 μg/cm²) of C-274, 333 pmol/cm² (10 μg/cm²) of H-271 and a mixture of32 pmol/cm² (1 μg/cm²) of C-274 and 333 pmol/cm² (10 μg/cm²) of H-271,respectively. After pre-incubating 2 ml of a virus supernatantcontaining 1,000 cfu of PM5neo virus in respective plates at 37° C. for30 minutes, the plates were thoroughly washed with PBS. To each of theseplates was added 2 ml of DMEM medium containing 2,000 NIH/3T3 cells andincubated at 37° C. for 2 hours. Non adhered cells were collected bydecantation and cells adhered to the plate were collected by trypsintreatment to detach them from the plate. The cells were combined. Theresultant cell suspension was divided into two halves. One half portionwas cultured in DMEM and the other portion was cultured in DMEMcontaining G418 at a final concentration of 0.75 mg/ml. Both portionswere incubated at 37° C. for 10 days and the colonies appeared werecounted. By taking the ratio of the number of G418^(r) colonies relativeto that obtained in the medium without G418 as the gene transferefficiency, the results are shown in FIG. 4. In FIG. 4, the abscissaindicates the functional materials used and the ordinate indicates thegene transfer efficiency.

As shown in FIG. 4, when using the plate on which the mixture of C-274and H-271 (molar ratio=1:10) was immobilized, the infection efficiencywas significantly increased. No gene transfer was observed in C-274immobilized plate.

(4) Gene Transfer Using C277-CS1

In order to investigate the effect on the infection efficiency by usingC277-CS1 as a material having cell binding domain and immobilization ofa mixture thereof and a material having retrovirus binding domain, thefollowing experiment was carried out. As the material binding to aretrovirus, a polylysine [(Lys)_(n), poly-L-lysyine hydrobromide,molecular weight: 50,000-100,000, Wako Pure Chemical Co., Ltd.] andH-271 were used. As the cells, non-adherent cells, TF-1 cells (ATCCCRL-2003), were used. First, according to the same manner as describedin Example 2 (9), immobilization on plates was carried out by using thefollowing solutions: C-277-CS1 (33 pmol/cm², 1.1 μg/cm²), polylysine(133 pmol/cm², 10 μg/cm²), a mixture of C-277-CS1 (33 pmol/cm²) andpolylysine (133 pmol/cm²), H-271 (333 pmol/cm², 10 μg/cm²) and a mixtureof C-277-CS1 (33 pmol/cm²) and H-271 (333 pmol/cm²) and CH-296 (33pmol/cm², 2.1 μg/cm²), respectively. To each plate was added RPMI 1640medium [containing 5 ng/ml of GM-CFS (Petro Tech), 50 units/ml ofpenicillin and 50 μg/ml of streptomycin] containing 1×10⁴ cfu of TKNEOvirus, 1×10⁴ of TF-1 cells and the plate was incubated at 37° C. for 24hours. After incubation, non adhered cells were collected by decantationand cells adhered to the plate were collected by trypsin treatment toremove them from the plate. The cells were combined. Respective onefifth portions of the resultant cell suspension were transferred to twoplates coated with CH-296 and incubated for 24 hours. Then, the mediumof one portion was exchanged to the above medium and that of the otherportion was exchanged to the above medium containing G418 at a finalconcentration of 0.75 mg/ml. Both portions were incubated at 37° C. for8 days and the colonies appeared were counted. The incidence of G418^(r)colonies (the gene transfer efficiency) was calculated based on thenumbers of colonies appeared in the presence and absence of G418.

The results are shown in FIG. 5. In FIG. 5, the abscissa indicates thefunctional materials used and the ordinate indicates the gene transferefficiency. In FIG. 5, (a) represents the use of the polylysine as theretrovirus binding material and (b) represents the use of H-271. Incomparison with the plate on which only retrovirus binding material wsaimmobilized, the gene transfer efficiency is significantly increased byusing the polylysine or H-271 together with C277-CS1 having the cellbinding domain.

(5) Preparation of Polypeptide Derived from Erythropoietin

For using in gene transfer into the cells having erythropoietinreceptor, a polypeptide derivative which was erythropoietin fused withglutathione-S-transferase (GST-Epo) was prepared. The amino acidsequence is shown in SEQ. ID No. 34 of the Sequence Listing. In thissequence, the amino acid sequence from 233rd amino acid to 398th aminoacid corresponds to erythropoietin.

First, a plasmid was constructed by the following procedures to expressGST-Epo. PCR was carried out by using cDNA library derived from humanfetal liver (Clonetech) as a template and primers EPF1 and EPR1 (thenucleotide sequences of primers EPF1 and EPR1 are shown in SEQ. ID Nos.35 and 36 of the Sequence Listing). A portion of the reaction mixturewas taken out and, by using it as a template and primers EPF2 and EPR2(the nucleotide sequences of primers EPF2 and EPR2 are shown in SEQ. IDNos. 37 and 38 of the Sequence Listing), additional PCR was carried out.Amplified DNA fragments were recovered from the reaction mixture,digested with EcoRI and BamHI (both Takara Shuzo Co., Ltd.) and thensubjected to agarose electrophoresis to recover a DNA fragment of about520 bp which contained a region encoding erythropoietin. The resultantfragment was mixed with a plasmid vector pTV118N (Takara Shuzo Co.,Ltd.) which digested with EcoRI (Takara Shuzo Co., Ltd.) and BamHI toligate it to the plasmid. Then, E. coli JM109 was transformed with theplasmid. A transformant maintaining the above plasmid was selected fromthe resultant transformants to prepare a plasmid and named as plasmidpEPO. Then, the plasmid pEPO thus obtained was digested with EcoRI andSalI (Takara Shuzo Co., Ltd.) and subjected to agarose electrophoresisto recover a DNA fragment of about 0.5 kb. This fragment was mixed witha plasmid vector pGEX5X-3 (Pharmacia) digested with EcoRI and SalI toligate them. E. coli JM109 was transformed with the resultant plasmid. Atransformant maintaining the above plasmid was selected from theresultant transformants to prepare a plasmid and the plasmid was namedas pGSTEPO. This plasmid encodes GST-EPO wherein the amino acid sequenceof erythropoietin is coupled to the C-terminal ofglutathione-S-transferase derived from the vector. The nucleotidesequence encoding GST-EPO on the plasmid pGSTEPO is shown in SEQ. ID No.39 of the Sequence Listing.

The polypeptide GST-Epo was prepared by the following procedures. Sevenculture tubes each containing 5 ml of LB broths containing 100 μg/ml ofampicillin were provided and E. coli JM109 transformed with the aboveplasmid pGSTEPO, Escherichia coli JM109/pGSTEPO, was inoculated intoeach broth, followed by incubating at 37° C. overnight. Then, seven 2liter Erlenmeyer flasks each containing 500 ml of the same broth wereprovided and 5 ml portions of the above culture were inoculated into theflasks, followed by incubating at 37° C. 3.5 Hours after startingincubation, IPTG was added at the final concentration of 1 mM andincubation was continued for additional 3.5 hours. After completion ofculture, cells were recovered from the culture broth by centrifugation,suspended in 100 ml of PBS containing 1 mM PMSF and 1 mM EDTA anddisrupted by sonication. To the disrupted solution was added 100 ml ofPBS containing 1 mM PMSF, 1 mM EDTA and 2% Triton X-100. The mixture wasallowed to stand on ice for 30 minutes and centrifuged to collect asupernatant. The resultant supernatant was filtered through a filter of0.45 μm (Millipore) and applied on a glutathione-Sephallose 4B column(Pharmacia, 3 ml) equilibrated with PBS. After washing the column withPBS, the column was eluted with 50 mM Tris-HCl containing 10 mMglutathione (pH 8.0). The eluate was analyzed by SDS-polyacrylamide gelelectrophoresis and a fraction containing a polypeptide having amolecular weight of about 44,000 was collected. The fraction wasdialyzed against PBS. A dialyzed sample was applied on Resource Q column(Pharmacia) equilibrated with PBS. After washing the column with PBS,the column was eluted with PBS having NaCl gradient of from 0 M to 0.6M. According to the same manner as described above, the column waseluted with 50 mM Tris-HCl containing glutathione (pH 8.0) to collect afraction containing a polypeptide having a molecular weight of about44,000. This was subjected to ultrafiltration with Centricon 10 (Amicon)to concentrate to about 50 μl. Further, it was filtrated with UltrafreeC3GVSTRL (Millipore) and the filtrate was subjected to gel filtrationchromatography using Superdex 200 column (Pharmacia, equilibrated withPBS). An eluted fraction containing a polypeptide having a molecularweight of about 44,000 was collected and this was used as a GST-Epopolypeptide solution in the subsequent experiments. In this GST-Eposolution, about 50% of the total proteins were GST-Epo.

(6) Gene Transfer into Erythropoietin Receptor Expressing Cells

The effect of gene transfer using erythropoietin as a material havingcell binding activity was investigated by using two kinds of cells, TF-1which expresses an erythropoietin receptor and HL-60 (ATCC CCL-240)which does not express the erythropoietin receptor. In thisinvestigation, the above polypeptide derivative of erythropoietin(GST-Epo) was used as erythropoietin and a polylysine was used as theretrovirus biding material. First, according to the same manner asdescribed in Example 2 (9), immobilization on plates was carried out byusing GST-Epo corresponding to 34 pmol/cm² (1.5 μg/cm²), polylysine (133pmol/cm², 10 μg/cm²), a mixture of GST-Epo (34 pmol/cm²) and polylysine(133 pmol/cm²), respectively. To each plate was added a mediumcontaining 1×10⁴ cfu of TKNEO virus and 1×10⁴ of cells and the plate wasincubated at 37° C. for 24 hours. As the medium, RPMI1640 medium(containing 5 ng/ml of GM-CFS, 50 units/ml of penicillin and 50 μg/ml ofstreptomycin) was used for TF-1 and RPMI medium (Nissui, containing 10%FCS, 50 units/ml of penicillin 50 μg/ml of streptomycin) was used forHL-60. After incubation, non adhered cells were collected by decantationand cells adhered to the plate were collected by trypsin treatment toremove them from the plate. The cells were combined. Respective onefifth portions of the resultant cell suspension were transferred to twoCH-296 immobilized plates and incubated for 24 hours. Then, the mediumof one portion was exchanged to the above medium and that of the otherportion was exchanged to the above medium containing G418 at a finalconcentration of 0.75 mg/ml. Both portions were incubated at 37° C. for8 days and the colonies appeared were counted. The incidence of G418^(r)colonies (the gene transfer efficiency) was calculated based on thenumbers of colonies appeared in the presence and absence of G418.

The results are shown in FIG. 6. In FIG. 6, the abscissa indicates thefunctional materials used and the ordinate indicates the gene transferefficiency, respectively. In case of using TF-1 cells as shown in FIG.6(a), although gene transfer was taken place to some extent in the plateon which only the polylysine was immobilized, the higher gene transferefficiency was obtained in the presence of GST-Epo. On the other hand,in case of using HL-60 as shown in FIG. 6(b), no increase in the genetransfer efficiency was observed in the presence of GST-Epo. Theseresults showed that target cell specific gene transfer was possible byusing erythropoietin.

In addition, an experiment of gene transfer into TF-1 cells was carriedout by replacing the retrovirus binding material with H2-547. Accordingto the same manner as described in Example 2 (9), immobilization onplates was carried out by using H2-547 (333 pmol/cm², 20 μg/cm²),GST-Epo corresponding to 34 pmol/cm², 1.5 μg/cm²) and a mixture ofGST-Epo (34 pmol/cm²) and H2-547 (333 pmol/cm², 20 μg/cm²),respectively. At the same time, a control experiment was carried out byusing BSA immobilized plate.

The results are shown in FIG. 7. In FIG. 7, the abscissa indicates thefunctional materials used and the ordinate indicates the gene transferefficiency, respectively. As shown in FIG. 7, in case of using H2-547,the gene transfer efficiency into TF-1 cells was increased in thepresence of GST-Epo.

(7) Gene Transfer Using Beads on which Mixture of Functional Materialswas Immobilized

Whether the retrovirus infection efficiency can be increased by usingbeads on which both material having cell binding domain and materialhaving retrovirus binding domain were immobilized or not wasinvestigated.

Beads on which polypeptides were immobilized were prepared according tothe following procedures. As beads, polystyrene beads having thediameter of 1.14 μm (Polybeads Polystyrene Microsphere, manufactured byPolyScience) were used. To 20 μl of a 2.5% suspension of the above beadswere added 80 μl of ethanol and 2 ml of various polypeptide solutions inPBS, followed by allowing to stand overnight at 4° C. To this were addedBSA and PBS to prepared 4 ml of 1% BSA/PBS suspension. Beads wererecovered from the suspension by centrifugation and suspended in 5 ml of1% BSA/PBS again. Then, the suspension was allowed to stand at roomtemperature for 1 hour to obtain a suspension of polypeptide immobilizedbeads. As the polypeptide solutions, 100 μg/ml of C-274, 100 μg/ml ofH-271, 100 μg/ml of CH-271, 100 μg/ml of CH-296 and a mixture of 100μg/ml of H-271 and 10 μg/ml of C-274. As a control, beads coated with 2%BSA solution was prepared according to the same manner.

One tenth portion of the polypeptide immobilized beads thus prepared wasrecovered from the above suspension and incubated at 37° C. overnighttogether with 2,000 of TF-1 cells and 1,000 cfu of TKNEO virussupernatant, respectively. The cells were recovered and suspended inRPMI medium [containing 10% of FCS, 5 ng/ml of GM-CFS (Petrotech), 50units/ml of penicillin and 50 μg/ml of streptomycin] containing 0.3% ofBacto agar (Difco) and seeded on a 35 mm plate made of the above mediumcontaining 0.5% of Bacto agar. Two mediums containing 0.75 mg/ml of G418and without G418 were used. The plate was incubated in 5% CO₂ at 37° C.for 14 days. Colonies which appeared in the presence of G418 and in theabsence of G418 were counted and the appearance ration of G418^(r)colonies (gene transfer efficiency) was calculated.

The results are shown in FIG. 8. In FIG. 8, the abscissa indicates thefunctional material used and BSA and the ordinate indicates the genetransfer efficiency. When using the beads on which the mixture of H-271and C-274 was immobilized, the higher gene transfer efficiency wasobtained in comparison with using beads on which only H-271 alone wasimmobilized and beads on which immobilized with CH-271 or CH-296 havingthe retrovirus binding domain and cell binding domain on the samemolecule, respectively.

EXAMPLE 4

(1) Gene Transfer Using FGF and C-FGF•A

The effect of FGF (Becton Deckinson) and the polypeptide represented bySEQ. ID No. 4 (C-FGF•A) on retrovirus infection was investigated byNIH/3T3 cell colony forming assay. Namely, assessment was carried outaccording to the same manner as described in Example 2 (9) byimmobilizing FGF (132 pmol/cm², 2.25 μg/cm²) and C-FGF•A (133 pmol/cm²,6.3 μg/cm²) on plates, respectively, and immobilizing BSA on a controlplate. To each plate was added 2 ml of a virus supernatant containing1,000 cfu of PM5neo virus and pre-incubated at 37° C. for 30 minutes,followed by thoroughly washing with PBS. To this plate was added 2 ml ofDMEM medium containing 2,000 NIH/3T3 cells and incubated at 37° C. for24 hours, followed by incubation in a selection medium containing 0.75mg/ml of G418 for 10 days. Colonies were stained and counted. Theresults are shown in FIG. 9. In FIG. 9, the abscissa indicates thefunctional material used and the ordinate indicates the number ofG418^(r) colonies appeared.

As shown in FIG. 9, no colony appeared in the control plate coated onwhich BSA was immobilized. On the other hand, when using FGF and C-FGF•Aimmobilized plate, G418^(r) colonies were identified in both plates.This result shows that both FGF anc C-FGF• have retrovirus bindingdomain and that C-FGF•A wherein the cell binding domain polypeptide offibronectin was coupled showed superior gene transfer to FGF.

(2) Relation between Concentration of C-FGF•A and Gene TransferEfficiency

The gene transfer efficiencies were compared by using plates coated withvarious concentrations of C-FGF•A. Infection with retrovirus was carriedout according to the same procedures as those in Example 4 (1) exceptfor the use of a plate prepared with 0.521 pmol/cm² (0.0247 μg/cm²)-5.21pmol/cm² (0.247 μg/cm²) of C-FGF•A according to the method described inExample 2 (9), and a BSA immobilized plate (control plate). After virusinfection treatment, the non-adhered were collected by decantation andthe cells adhered to the plate were collected by trypsin treatment toremove them from the plate. The cells thus collected were combined. Theresulting cell suspension was divided into two halves, one half portionwas cultured with DMEM and the other was cultured with DMEM containingG418 at the final concentration of 0.75 mg/ml. Both portions wereincubated at 37° C. for 10 days and the number of colonies whichappeared was counted. A ratio of the number of G418^(r) coloniesrelative to that of colonies obtained on a medium containing no G418 wastaken as the gene transfer efficiency.

The results are shown in FIG. 10. In FIG. 10, the abscissa indicates theconcentration of C-FGF•A used for immobilization on the plate and theordinate indicates the gene transfer efficiency. The experiment resultof control plate was also plotted at the polypeptide concentration of 0μmol/cm². As shown in FIG. 10, the gene transfer efficiency wasconcentration-dependently increased as increase in the C-FGF•Aconcentration upon immobilization.

(3) Gene Transfer into HL-60 Cell

As regards the retrovirus infection of HL-60 cell (ATCC CCL-240) whichis a non-adherent cell, the effect of the presence of variouspolypeptides was investigated according to the following procedures.Namely, to each of plate prepared using 100 pmol/cm² of C-FGF•A (4.8μg/cm²) or C-FGF-CS1 (5.1 μg/cm²) according to the method of Example 2(9) and a control plate on which BSA was immobilized was added 2 ml ofRPMI medium (containing 10% of FCS, 50 units/ml of penicillin and 50μg/ml of streptomycin) containing 1×10⁴ cfu of TKNEO virus and 2,000cells of HL-60, followed by incubation at 37° C. for 24 hours. Afterincubation, the non-adhered cells were collected by decantation and thecells adhered to the plate were collected by pipetting and these cellswere combined. Each ½ portion of the resulting cell suspension wastransferred to a plate coated with CH-296, incubated for 24 hours andthe medium was exchanged with RPMI medium containing the finalconcentration of 0.75 mg/ml of G418. After incubation at 37° C. for 12days, the number of colonies which appeared was counted. The number ofG418^(r) colonies obtained by using each polypeptide is shown in FIG.11. In FIG. 11, the abscissa indicates the functional material and theordinate indicates the number of G418^(r) colonies, respectively.

As shown in FIG. 11, the number of G418^(r) colonies was remarkablyincreased when C-FGF•A or C-FGF-CS1 immobilized plate was used,indicating that these polypeptides promote the infection of HL-60 cellwith retrovirus.

(4) Gene Transfer into Mouse Bone Marrow Cells

For investigating the effect of FGF, C-FGF•A and C-FGF-CS1 on retrovirusinfection of mouse myeloid cells, the following experiment was carriedout.

150 mg/kg 5-fluorouracil (5-FU, Amlesco) was administeredintraperitoneally to mouse (C3H/HeJ), 6 to 8 weeks age, femur and tibiawere isolated 2 days after administration to collect bone marrow. Theresulting bone marrow was subjected to density gradient centrifugationusing Ficoll-Hypaque (density 1.0875 g/ml, Pharmacia) to obtain a lowdensity mononuclear cell fraction which was used as mouse bone marrowcells.

The mouse bone marrow cells were pre-stimulated prior to infection withretrovirus according to a method by Luskey et al. (Blood, 80, 396(1992)). Namely, the mouse bone marrow cells were added to α-MEM (Gibco)containing 20% of FCS, 100 units/ml of recombinant human interleukin-6(rhIL-6, Amgen), 100 ng/ml of recombinant mouse stem cell factor (rmSCF,Amgen), 50 units/ml of penicillin and 50 μg/ml of streptomycin at celldensity of 1×10⁶ cells/ml, followed by incubation at 37° C. for 48 hoursin 5% CO₂. The pre-stimulated cells including those adhered to thecontainer were collected by aspiration with a pipette.

Each 2 ml of the medium, used for the above pre-stimulation, containing1×10⁶ pre-stimulated cells and 1×10⁴ cfu of PM5neo virus was added tothe plate prepared with 236 pmol/cm² (4 μg/cm²) of FGF, 169 pmol/cm² (8μg/cm²) of C-FGF•A or 159 pmol/cm² (8 μg/cm²) of C-FGF-CS1 according tothe method described in Example 2 (9), and a BSA immobilized plate(control plate), followed by incubation at 37° C. After 2 hours, amedium (2 ml) containing the same amount of virus was freshly added toeach plate, followed by continuing incubation for 22 hours. Aftercompletion of incubation, the non-adhered cells were collected bydecantation and the cells adhered to the plate were collected using acell dissociation buffer (CDB, containing no enzymes, Gibco) and thesecells were combined and washed twice with the same buffer. The number ofthe cells was counted. The collected cells were subjected to HPP-CFC(High Proliferative Potential-Colony Forming Cells) assay.

HPP-CFC assay was carried out according to a method by Bradley et al.(Aust. J. Exp. Biol. Med. Sci., 44, 287-293 (1966)). As a medium,1%/0.66% layered soft agar medium with or without G418 at the finalconcentration of 1.5 mg/ml was used. Infected cells was added thereto at1×10⁴ cells/well, followed by incubation at 37° C. for 13 days in 10%CO₂. After completion of incubation, the colonies which appeared wereobserved with an inverted microscope and the number of high densitycolonies (having the diameter of not less than 0.5 mm) derived fromHPP-CFC was counted to calculate the incidence (gene transferefficiency) of G418^(r) colonies. The results are shown in FIG. 12. InFIG. 12, the abscissa indicates the functional material used and BSA andthe ordinate indicates the gene transfer efficiency.

As shown in FIG. 12, no G418^(r) colonies appeared in the plate coatedwith BSA as a control, while the G418^(r) colonies were obtained whenthe plates on which the above respective polypeptides were immobilizedwere used. The gene transfer efficiencies were increased in an order ofin FGF, C-FGF•A and C-FGF-CS1, suggesting that the presence of the celladhesion domain derived from fibronectin and CS-1 polypeptide which hasthe binding activity to cells domain increase the infection of bonemarrow cells with retrovirus.

(5) Relation between Concentration of C277-ColV Used for Immobilizationon Plate and Gene Transfer Efficiency

The gene transfer efficiencies were compared by using plates coated withvarious concentration of C277-ColV according to the followingprocedures. The plates were prepared according to the method describedin Example 2 (9) using 0.1 pmol/cm² (0.1 μg/cm²)−416 pmol/cm² (20μg/cm²) of C277-ColV. 2 ml of a virus supernatant containing 1,000 cfuof PM5neo virus was added to respective plates and pre-incubation wascarried out at 37° C. for 30 minutes, followed by washing thoroughlywith PBS. To this plate was added 2 ml of DMEM medium containing 2,000NIH/3T3 cells and the plate was incubated at 37° C. for 24 hours.

The non-adhered cells were collected by decantation and the cellsadhered to the plate were collected by trypsin treatment to detach themfrom the plate and these cells were combined. The resulting cellsuspension was divided into two halves and one half portion was culturedin DMEM and the other portion was incubated in DMEM containing G418 atthe final concentration of 0.75 mg/ml at 37° C. for 10 days and thenumber of the colonies appeared was counted. A ratio of the number ofG418^(r) colonies relative to that of colonies obtained in a mediumcontaining no G418 was taken as the gene transfer efficiency. Theresults are shown in FIG. 13. In FIG. 13, the abscissa indicates thefunctional material used and the ordinate indicates the gene transferefficiency.

As shown in FIG. 13, when C277-ColV immobilized plate was used, the genetransfer efficiency was increased depending upon the concentration ofC277-ColV used for immobilization.

(6) Gene Transfer Using Polylysine

Binding of a polylysine [(Lys)_(n)] to a retrovirus was investigated bythe following procedures. As a polylysine, poly-L-lysyine hydrobromide(molecular weight: 50,000-100,000, Wako Pure Chemical) was used andaccording to the same manner as described in Example 2 (9), it wasimmobilized on a plate by using 133 pmol/cm² (10 μg/cm²) polylysinesolution in PBS. The gene transfer efficiencies of this plate and acontrol plate on which BSA was immobilized was assessed according to thesame manner as described in Example 4 (2). The results are shown in FIG.14. In FIG. 14, the abscissa indicates the functional material and theordinate indicates the gene transfer efficiency. As shown in FIG. 14, nocolony appeared in the control plate coated with BSA, while G418^(r)colonies appeared in the polylysine immobilized plate, suggesting that,after washing, the retrovirus remained on the plate because of bindingof the retrovirus to the polylysine immobilized on the plate.

EXAMPLE 5

(1) Gene Transfer Using Polymer of Polypeptide

The gene transfer using a polymer of a polypeptide was carried outwithout immobilization of the polypeptide on a plate. To a platepre-coated with BSA according to the method described in Example 2 (9)was added each 2 ml of DMEM containing 1,000 cfu of PM5neo virus, 2,000cells of NIH/3T3 cell and respective polypeptides (H-271, CH-271, H2-547and CH2-826) at the final concentration of 0.63 nmol/ml, followed byincubation for 24 hours. The non-adhered cells were collected bydecantation and the cells adhered to the plate were collected by trypsintreatment to remove them from the plate. Then, these cells werecombined. As a control, the same gene transfer experiment withoutaddition of any polypeptide was carried out according to the samemanner. The resulting cell suspension was divided into two halves andone half portion was cultured in DMEM. The other portion was cultured inDMEM containing G418 at the final concentration of 0.75 mg/ml. Bothportions were incubated at 37° C. for 10 days and colonies appeared werecounted. By taking the ratio of the number of G418^(r) colonies relativeto the number of colonies appeared in the medium without G418 as thegene transfer efficiency, the results are shown in FIG. 15. In FIG. 15,the abscissa indicates the functional material used and the ordinateindicates the gene transfer efficiency.

As seen from FIG. 15, the gene transfer efficiency in the presence ofH2-547 was significantly higher than that in the presence of H-271 and,in case of CH2-826, the gene transfer efficiency equal to or higher thanthat of CH-271 was obtained.

Further, more detailed investigation was carried out according to thesame manner as described above except that CH-271, CH-296 and H2-547were used as polypeptides in both amounts of 0.126 nmol (the finalconcentration of 0.063 nmol/ml) and 1.26 nmol (the final concentrationof 0.63 nmol/ml) for respective plates. The results are shown in FIG.16. In FIG. 16, the abscissa indicates the functional materials used andtheir amounts and the ordinate indicates the gene transfer efficiency.

As shown in from FIG. 16, when H2-547 was used, the gene transferefficiency was significantly higher than those of CH-271 and CH-296 ineither amount of the polypeptide.

(2) Gene Transfer into Mouse Bone Marrow Cells Using H2S-573

For investigation of the effect of H2S-573 on retrovirus infection ofbone marrow cells, an experiment of gene transfer into mouse bone marrowcells was carried out according to the same manner as described inExample 4 (4).

Mouse bone marrow cells were prepared according to the same manner asdescribed in the above Example and the cells were pre-stimulated.

As plates for retrovirus infection, in addition to H2S-573 (160pmol/cm², 10 μg/cm²) immobilized plate, CH-296 (132 pmol/cm², 8.3μg/cm²) immobilized plate and, as a control, BSA immobilized plate wereused. The results obtained by HPP-CFC assay are shown in FIG. 17. InFIG. 17, the abscissa indicates the functional material used and theordinate indicates the gene transfer efficiency.

As shown in FIG. 17, no high density colonies of G418^(r) appeared inthe plate coated with BSA as a control. Although about 50% of the genetransfer efficiency was obtained in CH-296 immobilized plate, highdensity colonies of G418^(r) were obtained at the higher efficiency incase of using H2S-573 immobilized plate.

EXAMPLE 6

(1) Gene Transfer Using Functional Material without Immobilization

The effect on the retrovirus infection efficiency when a polypeptide waspresent on a plate without immobilization was investigated as follows.Namely, to a plate pre-coated with BSA according to the method describedin Example 2 (9) was added each 2 ml of DMEM medium containing 100 cfuof PM5neo virus, 2,000 cells of NIH/3T3 cell and CH-296 at the finalconcentration of 10, 40, 250 μg/ml (each corresponding to 0.158, 0.632and 3.950 nmol/ml), followed by incubation for 24 hours. The non-adheredcells were collected by decantation and the cells adhered to the platewere collected by trypsin treatment to remove them from the plate. Thesecells were combined. The resulting cell suspension was transferred to a10 cm cell culture plate, followed by incubation for 24 hours. Themedium was exchanged with DMEM containing G418 at the finalconcentration of 0.75 mg/ml, followed by incubation for additional 10days. Separately, as a control, a plate without CH-296, and a plate onwhich 32 pmol/cm² (2 μg/cm²) or 127 pmol/cm² (8 μg/cm²) of CH-296 wasimmobilized were prepared and the above procedures were carried out byadding a virus supernatant and cells thereto. The number of G418^(r)colonies thus obtained was counted and the results are summarized inTable 1.

TABLE 1 Plate CH-296 Number of G418^(r) colonies BSA —  5 BSA  10 μg/ml41 BSA  40 μg/ml 66 BSA 250 μg/ml 92 CH-296 (32 pmol/cm²) — 55 CH-296(127 pmol/cm²) — 47

As shown in Table 1, when cell, virus and CH-296 were present togetherin the solution, the number of G418^(r) colonies was considerablyincreased in comparison with the absence of CH-296. The number was equalto or higher than that obtained by the use of the plate coated withCH-296. In addition, when a CH-296 solution was added, at the aboverespective concentrations, to a plate coated with BSA and, afterallowing to stand for a while, the plate was washed and used for virusinfection experiment, the number of G418^(r) colonies obtained wassimilar to that in the case without addition of CH-296 was obtained.From this, it is understood that CH-296 does not bind to a BSAimmobilized. Therefore, it is considered that the above retrovirusinfection promoting effect by CH-296 is not due to the adhesion ofCH-296 in the solution to a plate during incubation.

(2) Gene Transfer Using Functional Material without Immobilization

The effect on the retrovirus infection efficiency when polypeptides werepresent together on a plate without immobilization was investigated asfollows. Namely, to a plate pre-coated with BSA according to the methoddescribed in Example 2 (9) was added each 2 ml of DMEM medium containing1,000 cfu of PM5neo virus, 2,000 cells of NIH/3T3 cell, and C-FGF•A,ColV and C277-ColV at the final concentration of 1.67 nmol/ml,respectively, followed by incubation at 37° C. for 24 hours. Thenon-adhered cells were collected by decantation and the cells adhered tothe plate were collected by trypsin treatment to remove them from theplate. These cells were combined. The resulting cell suspension wasdivided into two halves, one half portion was cultured with DMEM and theother portion was cultured with DMEM containing G418 at the finalconcentration of 0.75 mg/ml. Both portions were incubated at 37° C. for10 days and the number of colonies which appeared was counted. A ratioof the number of G418^(r) colonies relative to that of colonies obtainedon a medium containing no G418 was taken as the gene transferefficiency. The results are shown in FIG. 18. In FIG. 18, the abscissaindicates the functional materials used and the ordinate indicates thegene transfer efficiency.

As shown in FIG. 18, when virus infection is taken place in the presenceof each polypeptide, the higher gene transfer efficiency is obtained.Thus, it is clear that, even when these polypeptides are not immobilizedon plates, the retrovirus infection is promoted.

(3) Gene Transduction of Non-Adherent Cells by Using Functional Materialwithout Immobilization

The effect on the gene transfer efficiency into non-adherent cells by apolypeptide without immobilization was investigated as follows. Namely,to each of a plate prepared with 333 pmol/cm² (10 μg/cm²) of H-271 andaccording to the same manner as that described in Example 2 (9) and acontrol plate on which BSA was immobilized was added 2 ml of RPMI medium(containing 5 ng/ml of GM-CFS, 50 units/ml of penicillin and 50 μg/ml ofstreptomycin) containing 1×10⁴ cfu of TKNE0 virus and 1×10⁴ cells ofTF-1 cells. To the BSA immobilized plate was further added H-271 at thefinal concentration of 50 μg/ml (1.67 nmol/ml) of H-271. Each plate wasincubated at 37° C. for 24 hours. After incubation, the non-adheredcells were collected by decantation and the cells adhered to the platewere collected by trypsin treatment. These cells were combined. Each ⅕portion of the resulting cell suspension was transferred to two platescoated with CH-296, incubated for 24 hours. The medium of one plate wasexchanged with the above medium and the medium of the other plate wasexchanged with the above medium containing G418 at the finalconcentration of 0.75 mg/ml. After incubation at 37° C. for 8 days, thenumber of colonies which appeared was counted. The incidence (genetransfer efficiency) was calculated based on the number of coloniesappeared in the presence and absence of G418. The results are shown inFIG. 19. In FIG. 19, the abscissa indicates the functional material andits form used and the ordinate indicates the gene transfer efficiency.

As shown in FIG. 19, when non-immobilized H-271 is used, the genetransfer efficiency obtained is higher than that obtained by usingimmobilized H-271. Then, it has been shown that, when using H-271 forgene transduction of TF-1 cells, a non-immobilized state is preferred.

(4) Elucidation of Mechanism of Retrovirus Infection Promotion byPolypeptide

In order to ascertain that promotion of retrovirus infection to cells bythe polypeptide without immobilization as shown in the above Examplesresulted from binding of the cells to the polypeptide and binding of thepolypeptide to the retrovirus, the following experiment was carried out.First, to BSA immobilized plates prepared with the method described inExample 2 (9) were added 2 ml of DMEM containing 1,000 cells of NIH/3T3cell, followed by incubation at 37° C. for 24 hours. The medium wasremoved from the plates, each 2 ml of 1.67 nmol/ml of H-271, CH-271,C-FGF•A and PBS as a control was added thereto, respectively, followedby incubation at 37° C. for 2.5 hours. The plates were washed with aHanks' balanced salt solution (HBSS, Gibco) containing 25 mM of HEPES. 2ml of a virus supernatant containing 1,000 cfu of PM5neo virus was addedto the plates, followed by incubation at 37° C. for 30 minutes. Theplates were washed with PBS. To these plates were added 2 ml of DMEM,followed by incubation at 37° C. for 24 hours. The non-adhered cellswere collected by decantation and the cells adhered to the plate werecollected by trypsin treatment to detach them from the plate. Thesecells were combined, respectively. Each cell suspension thus obtainedwas divided into two halves, one half portion was cultured with DMEM andthe other portion was cultured with DMEM containing G418 at the finalconcentration of 0.75 mg/ml. Both portions were incubated at 37° C. for10 days and the number of colonies which appeared was counted. A ratioof the number of G418^(r) colonies relative to that of colonies obtainedon a medium containing no G418 was taken as the gene transferefficiency. The results are shown in FIG. 20. In FIG. 20, the abscissaindicates the functional materials used and a control the ordinateindicates the gene transfer efficiency.

As shown in FIG. 20, when virus infection was carried out aftertreatment of the cells on the plate with the above polypeptide solution,the remarkable increase in the infection efficiency was observed. Thissuggests that the infection efficiency is increased by binding of thepolypeptide to cells and further binding of the retrovirus to thepolypeptide on the cells.

The similar experiment was carried out except that the polypeptide to beadded was replaced with 0.29 nmol/ml of C-FGF•A and 0.79 nmol/ml ofCH-296, respectively. The results are shown in FIG. 21. In FIG. 21, theabscissa indicates the functional materials used and a control theordinate indicates the gene transfer efficiency. As shown in FIG. 21,the increase in the gene transfer efficiency was observed in the case ofC-FGF•A and CH-296. Thus, the above activity was confirmed on C-FGF•A.At the same time, it was shown that CH-296 has the same activity topromote the retrovirus infection by the same mechanism.

EXAMPLE 7

(1) Gene Transfer Using Functional Material Immobilized on Beads

Whether the retrovirus infection efficiency could be increased by usingbeads coated with the functional material or not was investigatedaccording to the following procedures. As beads, polystyrene beadshaving the diameter of 1.14 μm (Polybeads Polystyrene Microsphere,manufactured by PolyScience) were used. To 20 μl of a 2.5% suspension ofthe above beads was added 80 μl of ethanol, and 2 ml of 40 μg/ml ofCH-296 was added thereto, followed by allowing to stand overnight at 4°C. To this were added BSA and PBS to prepare a 1% BSA/PBS suspension (4ml), beads were recovered by centrifugation and 5 ml of a 1% BSA/PBSsuspension was prepared again to allow to stand at room temperature for1 hour to obtain a suspension of CH-296 immobilized beads. As a control,beads were prepared according to the same manner except that theimmobilization was carried out by using 2% BSA instead of the CH-296solution.

One tenth portion (0.5 ml) was taken from the above bead suspension andthe beads were recovered by centrifugation. DMEM containing 1,000 cfu ofPM5neo virus was added thereto, followed by incubation at 37° C. for 30minutes. The beads were washed twice with 1% BSA/PBS, suspended in 2 mlof DMEM and 1 ml of which was transferred to a plate. 1 ml of DMEMcontaining 3×10⁵ cells of NIH/3T3 cell was added thereto, followed byincubation in CO₂ incubator at 37° C. for 24 hours. Thereafter, themedium was exchanged with DMEM containing G418 at the finalconcentration of 0.75 mg/ml, followed by incubation for another 10 days.Colonies which appeared were stained and counted. The results are shownin Table 2.

As shown in Table 2, when beads coated with CH-296 were used, 264colonies of G418^(r) appeared, while no resistant colonies were obtainedin case of using the beads coated with BSA as a control. This suggeststhat even immobilization of CH-296 on beads has the effect forincreasing retrovirus infection efficiency as in case of immobilizationon a plate.

TABLE 2 Beads Number of G418^(r) colonies BSA immobilized (control)  0CH-296 immobilized 264

(2) Gene Transfer into Mouse Bone Marrow Cells Using Beads on whichFunctional Material was Immobilized

The possibility of increase in the retrovirus infection efficiency ofmouse bone marrow cells with beads coated with the functional materialwas investigated according to the following procedures.

The mouse bone marrow cells were prepared according to the same manneras described in Example 4 (4) and pre-stimulated.

Each 2 ml of the medium, used for the above pre-stimulation, containing1×10⁶ pre-stimulated cells and 1×10⁴ cfu of PM5neo virus was added to aplate coated with BSA according to the same manner as described inExample 2 (9) and the similar plate coated with BSA to which 1/10portion of the CH-296 immobilized beads as prepared in Example 7 (1),followed by incubation at 37° C. After 2 hours, a medium (2 ml)containing the same amount of virus was freshly added to each plate,followed by continuing incubation for 22 hours. After completion ofincubation, the non-adhered cells were collected by decantation and thecells adhered to the plate were collected using a cell dissociationbuffer (CDB, containing no enzymes, Gibco) and these cells were combinedand washed twice with the same buffer. The number of the cells wascounted. The collected cells were subjected to HPP-CFC assay accordingto the same manner as described in Example 4 (4).

The results are shown in FIG. 22. In FIG. 22, the abscissa indicates thefunctional material and its form used and the ordinate indicates thegene transfer efficiency. As shown in the results, it is understood thatthe retrovirus infection efficiency of mouse bone marrow cells can alsobe increase by using CH-296 immobilized beads.

EXAMPLE 8

(1) Gene Transfer Using H-271 and CH-271

The effects of H-271 on retrovirus infection was assessed bypre-incubating a virus supernatant in plates coated with H-271 andCH-271 which was known to promote retrovirus infection, respectively,after thoroughly washing the plates, determining the remaining amount ofthe virus by NIH/3T3 cell colony formation assay and comparing theresults of both plates. Namely, according to the same manner asdescribed in Example 2 (9), plates were prepared with variousconcentrations of H-271 [67 pmol/cm² (2 μg/cm²) to 333 pmol/cm² (10μg/cm²)] and CH-271 [67 pmol/cm² (4 μg/cm²) to 333 pmol/cm² (20μg/cm²)], respectively. To each plate was added 2 ml of a virussupernatant containing 1,000 cfu of PM5neo virus and pre-incubated at37° C. for 30 minutes, followed by thoroughly washing with PBS. To thisplate was added 2 ml of DMEM medium containing 2,000 NIH/3T3 cells andincubated at 37° C. for 24 hours, followed by incubation in a selectionmedium containing 0.75 mg/ml of G418 for 10 days. Colonies were stainedand counted. The results are shown in FIG. 23. FIG. 23 is a graphillustrating the relation between the functional material and the genetransfer efficiency. In FIG. 23, the abscissa indicates the amount ofthe functional material used and the ordinate indicates the number ofG418^(r) colonies.

As shown in FIG. 23, when using CH-271 immobilized plate, the number ofG418^(r) colonies appeared was almost the same regardless of theconcentration of the polypeptide. On the other hand, in case of H-271,the number of colonies appeared was increased depending upon theconcentration as increase in the concentration of the polypeptide usedin immobilization and, in case of the plate prepared with 333 pmol/cm²of H-271, the number of the colonies appeared was almost the same asthat of CH-271. This suggests that the equivalent virus infectionefficiency to that of CH-271 can be obtained, when a sufficient amountof H-271 is immobilized on a plate.

(2) Gene transfer Using C-FGF•A

The effects of C-FGF•A on retrovirus infection was investigated byNIH/3T3 cell colony assay. Namely, assessment was carried out accordingto the same manner as described in Example 8 (1) except for the use ofplates prepared with 127 pmol/cm² (6 μg/cm²) of C-FGF•A, 127 pmol/cm²(7.6 μg/cm²) of CH-271 and 127 pmol/cm² (8 μg/cm²) of CH-289 accordingto the method described in Example 2 (9) and a control plate on whichBSA was immobilized. The results are shown in FIG. 24. FIG. 24 is agraph illustrating the relation between the functional materials and thegene transfer efficiencies. In FIG. 24, the abscissa indicates thefunctional materials and BSA and the ordinate indicates the genetransfer efficiency.

As shown in FIG. 24, no colony appeared in the control plate on whichBSA was immobilized. On the other hand, when using the plate on whichC-FGF•A was immobilized, appearance of G418^(r) colonies was confirmedand the number of the colonies was the same as those of the plates usingCH-271 and CH-296. This suggests that a retrovirus binding domain havingsubstantially the same functions as those of CH-271 and CH-296 ispresent on FGF molecule.

(3) Gene Transfer Using C-FGF-CS1

The effects of C-FGF-CS1 polypeptide on retrovirus infection wasinvestigated according to the following procedures. Namely, NIH/3T3 cellcolony assay was carried out according to the same manner as describedin Example 8 (1) by using plates prepared with 133 pmol/cm² of C-FGF-CS1(6.7 μg/cm²), C-FGF•A (6.3 μg/cm²), CH-271 (8 μg/cm²), and CH-296 (8.4μg/cm²), respectively, according to the method described in Example 2(9). The results are shown in FIG. 25. FIG. 25 is a graph illustratingthe relation between the functional materials and the gene transferefficiencies. In FIG. 25, the abscissa indicates the functionalmaterials used and the ordinate indicates the number of G418^(r)colonies.

As shown in FIG. 25, almost the same number of colonies appear in theplates on which these four polypeptides were immobilized, respectively,indicating that C-FGF-CS1 molecule has the retrovirus binding activityequivalent to the other polypeptides.

(4) Gene Transfer Using C277-ColV

The effects of C277-ColV polypeptide on retrovirus infection wasassessed according to the same manner as in Example 8 (1) by using aplate prepared with 124 pmol/cm² (6.4 μg/cm²) of C277-ColV and a controlplate on which BSA was immobilized. The results are shown in FIG. 26.FIG. 26 is a graph illustrating the relation between the functionalmaterial and the gene transfer efficiency. The abscissa indicates thefunctional material used and BSA and the ordinate indicates the numberof G418^(r) colonies.

As shown in FIG. 26, no colony appeared in the control plate coated withBSA. On the other hand, when using C277-ColV immobilized plate, G418^(r)colonies appeared. This indicates that the retrovirus remains on theplate after washing due to the presence of a retrovirus binding domainon the ColV molecule.

As described hereinabove, the present invention provides a method forefficient gene transfer into target cells with retroviruses. When themethod of the present invention is carrying out by selecting a cellbinding material suitable for target cells, transformed target cells canbe obtained conveniently at the high gene transfer efficiency withoutany necessity of a special retrovirus vector. By grafting thetransformed cells into vertebrate, transformed animal is readilyprepared and the present invention is useful in various technical fieldssuch as medical sciences, cell technology, genetic engineering anddevelopmental technology. In addition, there are provided a culturemedium containing the functional material of the present invention or amixture thereof and a reagent kit for carrying out retrovirus mediatedgene transfer into target cells. By using these culture medium and kit,localization of a retrovirus, transduction of an exogenous gene intotarget cells and the like can be readily and efficiently carried out.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a graph illustrating the gene transfer efficiencies into thetarget cells with the fibroblast growth factor, the functional materialcontaining the fibroblast growth factor and the mixture of thefibroblast growth factor and the cell adhesion domain polypeptide offibronectin.

FIG. 2 is a graph illustrating the gene transfer efficiencies into thetarget cells with the fibroblast growth factor, the mixture of thefibroblast growth factor and the cell adhesion domain polypeptide offibronectin and the cell adhesion domain polypeptide of fibroncetin.

FIG. 3 is a graph illustrating the gene transfer efficiencies into thetarget cells with the collagen fragment, the mixture of the cell bindingdomain polypeptide of fibronectin and the collagen fragment, thefunctional material containing the collagen fragment and the mixture ofthe cell binding domain polypeptide of fibronectin.

FIG. 4 is a graph illustrating the gene transfer efficiencies into thetarget cells with the fibronectin fragment and the mixture of thefibronectin fragment and the cell binding domain polypeptide offibronectin.

FIG. 5 is a graph illustrating the gene transfer efficiencies into thetarget cells with the cell binding domain polypeptide of fibronectin,the polylysine, the mixture of the polylysine and the cell bindingdomain polypeptide of fibronectin, the fibronectin fragment and themixture of the fibronectin fragment and the cell binding domainpolypeptide of fibronectin.

FIG. 6 is a graph illustrating the gene transfer efficiencies into thetarget cells with the erythropoietin derivative, the polylysine and themixture of the erythropoietin derivative and the polylysine.

FIG. 7 is a graph illustrating the gene transfer efficiencies into thetarget cells with the erythropoietin derivative, the fibronectinfragment polymer and the mixture of the erythropoietin derivative andthe fibronectin fragment polymer.

FIG. 8 is a graph illustrating the gene transfer efficiencies into thetarget cells with the beads on which the fibronectin fragment wasimmobilized, the beads on which the cell binding domain polypeptide offibronectin was immobilized and the beads on which the mixture of thefibronectin fragment and the cell binding domain polypeptide offibronectin was immobilized.

FIG. 9 is a graph illustrating the transformation of the target cellswith the fibroblast growth factor and the functional material containingthe fibroblast growth factor.

FIG. 10 is a graph illustrating the relation between the amount of thefunctional material containing the fibroblast growth factor used and thegene transfer efficiency.

FIG. 11 is a graph illustrating the transformation of the target cellswith the functional material containing fibroblast growth factor.

FIG. 12 is another graph illustrating the transformation of the targetcells with the functional material containing the fibroblast growthfactor.

FIG. 13 is a graph illustrating the relation between gene transferefficiency into the target cells and the amount of the functionalmaterial containing the collagen fragment used.

FIG. 14 is a graph illustrating the gene transfer efficiency into thetarget cells with the polylysine.

FIG. 15 is a graph illustrating the transformation of the target cellswith the fibronectin fragment and the fibronectin fragment polymer.

FIG. 16 is another graph illustrating the transformation of target cellswith the fibronectin fragment and the fibronectin fragment polymer.

FIG. 17 is yet another graph illustrating the gene transfer efficiencyinto the target cells with the fibronectin fragment and the fibronectinfragment polymer.

FIG. 18 is a graph illustrating the gene transfer efficiency into thetarget cells with the functional material containing the fibroblastgrowth factor, the collagen fragment and the functional materialcontaining the collagen fragment.

FIG. 19 is a graph illustrating the gene transfer efficiency into thetarget cells with the fibronectin fragment.

FIG. 20 is a graph illustrating the gene transfer efficiency into thetarget cells with the functional material containing the fibronectinfragment and fibroblast growth factor.

FIG. 21 is a graph illustrating the gene transfer efficiency into thetarget cells with the functional material containing the fibroblastgrowth factor and the fibronectin fragment.

FIG. 22 is a graph illustrating the gene transfer efficiency into thetarget cells with the fibronectin fragment immobilized beads.

FIG. 23 is a graph illustrating the relation between the amount of thefibronectin fragment used and the gene transduction of the target cells.

FIG. 24 is a graph illustrating the gene transduction of the targetcells with the functional material containing the fibroblast growthfactor and the fibronectin fragment.

FIG. 25 is another graph illustrating the gene transduction of thetarget cells with the functional material containing the fibroblastgrowth factor and the fibronectin fragment.

FIG. 26 is a graph illustrating the gene transduction of the targetcells with the functional material containing the collagen fragment.

1. An isolated and purified polypeptide which facilitates binding of aretroviral vector to a target cell having CS-1 binding properties,wherein said retroviral vector is capable of transferring a gene intosaid target cell, and wherein said polypeptide binds to a retroviralvector and the target cell and consists essentially of: SEQ. ID No.5.